Genes expressed in prostate cancer
The present invention relates to a combination comprising a plurality of cDNAs which are differentially expressed in prostate cancer and which may be used in their entirety or in part as to diagnose, to stage to treat or to monitor the progression or treatment of prostate cancer.
[0001] This application claims the benefit of Provisional Application No. 60,295,048 filed May 31, 2001.
FIELD OF THE INVENTION[0002] The present invention relates to a combination comprising a plurality of cDNAs which are differentially expressed in prostate cancer and which may be used entirely or in part to diagnose, to stage, to treat, or to monitor the progression or treatment of prostate cancer.
BACKGROUND OF THE INVENTION[0003] Array technology can provide a simple way to explore the expression of a single polymorphic gene or the expression profile of a large number of related or unrelated genes. When the expression of a single gene is examined, arrays are employed to detect the expression of a specific gene or its variants. When an expression profile is examined, arrays provide a platform for examining which genes are tissue specific, carrying out housekeeping functions, parts of a signaling cascade, or specifically related to a particular genetic predisposition, condition, disease, or disorder.
[0004] The potential application of gene expression profiling is particularly relevant to improving diagnosis, prognosis, and treatment of disease. For example, both the levels and sequences expressed in tissues from subjects with prostate cancer may be compared with the levels and sequences expressed in normal tissue.
[0005] Prostate cancer is a common malignancy in men over the age of 50, and the incidence increases with age. In the US, there are approximately 132,000 newly diagnosed cases of prostate cancer and more than 33,000 deaths from the disorder each year.
[0006] Once cancer cells arise in the prostate, they are stimulated by testosterone to a more rapid growth. Thus, removal of the testes can indirectly reduce both rapid growth and metastasis of the cancer. Over 95 percent of prostatic cancers are adenocarcinomas which originate in the prostatic acini. The remaining 5 percent are divided between squamous cell and transitional cell carcinomas, both of which arise in the prostatic ducts or other parts of the prostate gland.
[0007] As with most cancers, prostate cancer develops through a multistage progression ultimately resulting in an aggressive, metastatic phenotype. The initial step in tumor progression involves the hyperproliferation of normal luminal and/or basal epithelial cells that become hyperplastic and evolve into early-stage tumors. The early-stage tumors are localized in the prostate but eventually may metastasize, particularly to the bone, brain or lung. About 80% of these tumors remain responsive to androgen treatment, an important hormone controlling the growth of prostate epithelial cells. However, in its most advanced state, cancer growth becomes androgen-independent and there is currently no known treatment for this condition.
[0008] A primary diagnostic marker for prostate cancer is prostate specific antigen (PSA). PSA is a tissue-specific serine protease almost exclusively produced by prostatic epithelial cells. The quantity of PSA correlates with the number and volume of the prostatic epithelial cells, and consequently, the levels of PSA are an excellent indicator of abnormal prostate growth. Men with prostate cancer exhibit an early linear increase in PSA levels followed by an exponential increase prior to diagnosis. However, since PSA levels are also influenced by factors such as inflammation, androgen and other growth factors, some scientists maintain that changes in PSA levels are not useful in detecting individual cases of prostate cancer.
[0009] Current areas of cancer research provide additional prospects for markers as well as potential therapeutic targets for prostate cancer. Several growth factors have been shown to play a critical role in tumor development, growth, and progression. The growth factors Epidermal Growth Factor (EGF), Fibroblast Growth Factor (FGF), and Tumor Growth Factor alpha (TGF&agr;) are important in the growth of normal as well as hyperproliferative prostate epithelial cells, particularly at early stages of tumor development and progression, and affect signaling pathways in these cells in various ways (Lin et al. (1999) Cancer Res 59:2891-2897; Putz et al. (1999) Cancer Res 59:227-233). The TGF-&bgr; family of growth factors are generally expressed at increased levels in human cancers and the high expression levels in many cases correlates with advanced stages of malignancy and poor survival (Gold (1999) Crit Rev Oncog 10:303-360). Finally, there are human cell lines representing both the androgen-dependent stage of prostate cancer (LNCap) as well as the androgen-independent, hormone refractory stage of the disease (PC3 and DU-145) that have proved useful in studying gene expression patterns associated with the progression of prostate cancer, and the effects of cell treatments on these expressed genes (Chung (1999) Prostate 15:199-207).
[0010] The present invention provides a combination comprising a plurality of cDNAs and individual cDNAs that can be employed for the diagnosis, prognosis or treatment of prostate cancer. The present invention satisfies a need in the art in that it provides a set of differentially expressed cDNAs which may be used entirely or in part to diagnose, to stage, to treat, or to monitor the progression or treatment of a subject with prostate cancer.
SUMMARY[0011] The present invention provides a combination comprising a plurality of cDNAs and their complements which are expressed in prostate cancer and which are selected from SEQ ID NOs:1-501 as presented in the Sequence Listing. In one embodiment, each cDNA, specifically SEQ ID NOs: 1-56, 87-153, 164-349, 370-414, and 437-501, is downregulated at least two-fold; in another embodiment, each cDNA, specifically SEQ ID NOs:57-86, 154-163, 350-369, and 415-436, is upregulated at least two-fold. In one aspect, the combination is useful to diagnose or treat prostate cancer. In another aspect, the combination is immobilized on a substrate.
[0012] The invention also provides an isolated cDNA selected from SEQ ID NOs:14, 26, 40, 52, 55, 60, 65, 68, 73, 79, 82, 85, 92, 110, 112, 114, 115, 117, 122, 125, 126, 130, 136, 137, 139, 141, 143, 144, 145, 146, 147, 160, 164, 166, 167, 168, 190, 191, 194, 195, 199, 201, 204, 211, 212, 222, 224, 226, 229, 233, 234, 240, 243, 245, 248, 250, 253, 254, 259, 264, 268, 269, 270, 272, 276, 278, 279, 281, 282, 284, 285, 286, 293, 296, 297, 299, 300, 301, 302, 306, 308, 312, 313, 314, 317, 319, 321, 321, 322, 322, 323, 324, 325, 326, 330, 331, 332, 334, 336, 337, 338, 339, 340, 342, 346, 353, 355, 357, 361, 365, 366, 371, 372, 376, 380, 383, 385, 386, 387, 390, 399, 400, 402, 405, 406, 408, 409, 410, 412, 413, 417, 418, 419, 420, 422, 422, 424, 426, 438, 444, 445, 453, 456, 460, 461, 471, 479, 480, 487, 490, 492, 495, 496, and 497 as presented in the Sequence Listing. The invention also provides a vector comprising the cDNA, a host cell comprising the vector, and a method for producing a protein comprising culturing the host cell under conditions for the expression of a protein and recovering the protein from the host cell culture.
[0013] The invention further provides a method to detect differential expression of one or more of the cDNAs of the combination, the method comprising: hybridizing the substrate comprising the combination with the nucleic acids of a sample, thereby forming one or more hybridization complexes, detecting the hybridization complexes, and comparing the hybridization complexes with those of a standard, wherein differences in the size and signal intensity of each hybridization complex indicates differential expression of nucleic acids in the sample. In one aspect, the sample is from prostate, and differential expression determines an early, mid, and late stage of the disorder.
[0014] The invention still further provides a method of screening a library or a plurality of molecules or compounds to identify a ligand, the method comprising: combining the substrate comprising the combination with a library or a plurality of molecules or compounds under conditions to allow specific binding and detecting specific binding, thereby identifying a ligand. The library or a plurality of molecules or compounds are selected from DNA molecules, RNA molecules, peptide nucleic acid molecules, mimetics, peptides, transcription factors, repressors, and other regulatory proteins. The invention additionally provides a method for purifying a ligand, the method comprising combining a cDNA of the invention with a sample under conditions which allow specific binding, recovering the bound cDNA, and separating the cDNA from the ligand, thereby obtaining purified ligand.
[0015] The present invention provides a purified protein encoded and produced by a cDNA of the invention. The invention also provides a method for using a protein to screen a library or a plurality of molecules or compounds to identify a ligand, the method comprising: combining the protein or a portion thereof with the library or a plurality of molecules or compounds under conditions to allow specific binding and detecting specific binding, thereby identifying a ligand which specifically binds the protein. A library or a plurality of molecules or compounds are selected from DNA molecules, RNA molecules, peptide nucleic acid molecules, mimetics, peptides, proteins, agonists, antagonists, antibodies or their fragments, immunoglobulins, inhibitors, drug compounds, and pharmaceutical agents. The invention further provides for using a protein to purify a ligand, the method comprising: combining the protein or a portion thereof with a sample under conditions to allow specific binding, recovering the bound protein, and separating the protein from the ligand, thereby obtaining purified ligand. The invention still further provides a composition comprising the protein and a pharmaceutical carrier. The invention yet still further provides a method for using the protein to produce an antibody, the method comprising: immunizing an animal with the protein or an antigenic determinant under conditions to elicit an antibody response, isolating animal antibodies, and screening the isolated antibodies with the protein to identify an antibody which specifically binds the protein. The invention even further provides a method for using the protein to purify antibodies which bind specifically to the protein.
[0016] The present invention provides a purified antibody. The invention also provides a method of using an antibody to detect the expression of a protein in a sample, the method comprising contacting the antibody with a sample under conditions for the formation of an antibody:protein complex and detecting complex formation wherein the formation of the complex indicates the expression of the protein in the sample. In one aspect, the sample is from prostate. In another aspect, complex formation is compared to standards and is diagnostic of prostate cancer. The invention further provides using an antibody to immunopurify a protein comprising combining the antibody with a sample under conditions to allow formation of an antibody:protein complex, and separating the antibody from the protein, thereby obtaining purified protein. The invention still further provides a method of using an antibody to detect prostate cancer, the method comprises contacting a sample with the antibody which specifically binds a protein of the invention under conditions to form an antibody:protein complex, detecting antibody:protein complex formation, and comparing complex formation with standards, wherein complex formation indicates the presence of prostate cancer in the sample.
[0017] The invention provides a composition comprising a cDNA, a protein, an antibody, or a ligand with agonistic or antagonistic activity that can be used in the methods of the invention or to treat prostate cancer.
DESCRIPTION OF THE COMPACT DISC-RECORDABLE (CD-R)[0018] CD-R 1 is labeled: “PA-0027-1 US, Copy 1,” was created on May 29, 2002 and contains: the Sequence Listing formatted in plain ASCII text. The file for the Sequence Listing is entitled pa00271us_seqlist.txt, created on May 29, 2002 and is 1,430 KB in size.
[0019] CD-R 2 is an exact copy of CD-R 1. CD-R 2 is labeled: “PA-0027-1 US, Copy 2,” and was created on May 29, 2002.
[0020] The CD-R labeled as: “PA-0027-1 US, CRF,” contains the Sequence Listing formatted in plain ASCII text. The file for the Sequence Listing is entitled pa00291us_seqlist.txt, was created on May 29, 2002 and is 1,430 KB in size.
[0021] The content of the Sequence Listing named above and as described below, submitted in duplicate on two (2) CD-Rs (labeled “PA-0027-1 US, Copy 1” and “PA-0027-1 US, Copy 2”), and the CRF (labeled “PA-0027-1 US, CRF”) containing the Sequence Listing, are incorporated by reference herein, in their entirety.
DESCRIPTION OF THE SEQUENCE LISTING AND TABLES[0022] A portion of the disclosure of this patent document contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure, as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.
[0023] The Sequence Listing is a compilation of cDNAs obtained by sequencing and extension of clone inserts. Each sequence is identified by a sequence identification number (SEQ ID NO) and by the template number (TEMPLATE ID) from which it was obtained.
[0024] Table 1 lists the functional annotation and differential expression of the cDNAs of the present invention. Columns 1 and 2 show the SEQ ID NO and TEMPLATE ID, respectively. Columns 3, 4, and 5 show the GenBank hit (GI Number), probability score (E-value), and functional annotation, respectively, as determined by BLAST analysis (version 1.4 using default parameters; Altschul (1993) J Mol Evol 36:290-300; Altschul et al. (1990) J Mol Biol 215:403-410) of the cDNA against GenBank (release 116; National Center for Biotechnology Information (NCBI), Bethesda Md.). Column 6 shows the prostate cancer cell line in which differential expression was observed. The table is further subdivided by the treatment group and up- or down-regulated expression.
[0025] Table 2 shows Pfam annotations for proteins encoded by the cDNAs of the present invention. Columns 1 and 2 show the SEQ ID NO and TEMPLATE ID of each cDNA, respectively. Columns 3, 4, and 5 show the first nucleotide (START), last nucleotide (STOP), and reading frame, respectively, for the protein encoded by the cDNA as identified by Pfam analysis of the encoded protein. Columns 6 and 7 show the Pfam description and E-values, respectively, corresponding to the protein domain encoded by the cDNA.
[0026] Table 3 shows signal peptide and transmembrane motifs predicted for the protein encoded by the cDNAs of the present invention. Columns 1 and 2 show the SEQ ID NO and TEMPLATE ID of each cDNA, respectively. Columns 3, 4, and 5 show the first nucleotide (START), last nucleotide (STOP), and reading frame, respectively, for the protein encoded by the cDNA, and column 6 identifies the signal peptide (SP) or transmembrane (TM) domain for the encoded protein.
[0027] Table 4 shows the region of each cDNA encompassed by the clone present on a microarray and identified as differentially expressed. Columns 1 and 2 show the SEQ ID NO and TEMPLATE ID of each cDNA, respectively. Column 3 shows the CLONE ID and columns 4 and 5 show the first nucleotide (START) and last nucleotide (STOP) encompassed by the clone on the template.
DESCRIPTION OF THE INVENTION[0028] Definitions “Antibody” refers to intact immunoglobulin molecule, a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a recombinant antibody, a humanized antibody, single chain antibodies, a Fab fragment, an F(ab′)2 fragment, an Fv fragment, and an antibody-peptide fusion protein. “Antigenic determinant” refers to an antigenic or immunogenic epitope, structural feature, or region of an oligopeptide, peptide, or protein which is capable of inducing formation of an antibody which specifically binds the protein. Biological activity is not a prerequisite for immunogenicity. “Array” refers to an ordered arrangement of at least two cDNAs, proteins, or antibodies on a substrate. At least one of the cDNAs, proteins, or antibodies represents a control or standard, and the other cDNA, protein, or antibody is of diagnostic or therapeutic interest. The arrangement of at least two and up to about 40,000 cDNAs, proteins, or antibodies on the substrate assures that the size and signal intensity of each labeled complex, formed between each cDNA and at least one nucleic acid, each protein and at least one ligand or antibody, or each antibody and at least one protein to which the antibody specifically binds, is individually distinguishable.
[0029] A “combination” refers to at least two and up to about 1002 cDNAs wherein the cDNAs are SEQ ID NOs: 1-501 as presented in the Sequence Listing and the complements thereof.
[0030] The “complement” of a cDNA of the Sequence Listing refers to a nucleic acid molecule which is completely complementary over its full length and which will hybridize to a nucleic acid molecule under conditions of high stringency. “cDNA” refers to an isolated polynucleotide, nucleic acid molecule, or any fragment thereof that contains from about 400 to about 12,000 nucleotides. It may have originated recombinantly or synthetically, may be double-stranded or single-stranded, may represent coding and noncoding 3′ or 5′ sequence, and generally lacks introns.
[0031] The phrase “cDNA encoding a protein” refers to a nucleic acid whose sequence closely aligns with sequences that encode conserved regions, motifs or domains identified by employing analyses well known in the art. These analyses include BLAST (Altschul, supra; Altschul et al., supra) and BLAST2 (Altschul et al. (1997) Nucleic Acids Res 25:3389-3402) which provide identity within the conserved region. Brenner et al. (1998; Proc Natl Acad Sci 95:6073-6078) who analyzed BLAST for its ability to identify structural homologs by sequence identity found 30% identity is a reliable threshold for sequence alignments of at least 150 residues and 40% is a reasonable threshold for alignments of at least 70 residues (Brenner, page 6076, column 2).
[0032] A “composition” refers to the polynucleotide and a labeling moiety; a purified protein and a pharmaceutical carrier or a heterologous, labeling or purification moiety; an antibody and a labeling moiety or pharmaceutical agent; and the like. “Derivative” refers to a cDNA or a protein that has been subjected to a chemical modification. Derivatization of a cDNA can involve substitution of a nontraditional base such as queosine or of an analog such as hypoxanthine. These substitutions are well known in the art. Derivatization of a protein involves the replacement of a hydrogen by an acetyl, acyl, alkyl, amino, formyl, or morpholino group. Derivative molecules retain the biological activities of the naturally occurring molecules but may confer longer lifespan or enhanced activity. “Differential expression” refers to an increased or upregulated or a decreased or downregulated expression as detected by absence, presence, or at least two-fold change in the amount of transcribed messenger RNA or translated protein in a sample. “Disorder” refers to neoplastic conditions and diseases such as cancer, and in particular, prostate cancer.
[0033] An “expression profile” is a representation of gene expression in a sample. A nucleic acid expression profile is produced using sequencing, hybridization, or amplification technologies using mRNAs or cDNAs from a sample. A protein expression profile, although time delayed, mirrors the nucleic acid expression profile and is produced using gel electrophoresis, mass spectrometry, or an array and labeling moieties or antibodies which specifically bind the protein. The nucleic acids, proteins, or antibodies specifically binding the protein may be used in solution or attached to a substrate. “Fragment” refers to a chain of consecutive nucleotides from about 50 to about 4000 base pairs in length. Fragments may be used in PCR or hybridization technologies to identify related nucleic acid molecules and in binding assays to screen for a ligand. Such ligands are useful as therapeutics to regulate replication, transcription or translation.
[0034] A “hybridization complex” is formed between a cDNA and a nucleic acid of a sample when the purines of one molecule hydrogen bond with the pyrimidines of the complementary molecule, e.g., 5′-A-G-T-C-3′ base pairs with 3′-T-C-A-G-5′. Hybridization conditions, degree of complementarity and the use of nucleotide analogs affect the efficiency and stringency of hybridization reactions. “Identity” as applied to sequences, refers to the quantification (usually percentage) of nucleotide or residue matches between at least two sequences aligned using a standardized algorithm such as Smith-Waterman alignment (Smith and Waterman (1981) J Mol Biol 147:195-197), CLUSTALW (Thompson et al. (1994) Nucleic Acids Res 22:4673-4680), or BLAST2 (Altschul (1997, supra). BLAST2 may be used in a standardized and reproducible way to insert gaps in one of the sequences in order to optimize alignment and to achieve a more meaningful comparison between them. “Similarity” uses the same algorithms but takes conservative substitution of residues into account. In proteins, similarity exceeds identity in that substitution of a valine for a leucine or isoleucine, is counted in calculating the reported percentage. Substitutions which are considered to be conservative are well known in the art. “Isolated or “purified” refers to any molecule or compound that is separated from its natural environment and is from about 60% free to about 90% free from other components with which it is naturally associated. “Labeling moiety” refers to any reporter molecule including radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, substrates, cofactors, inhibitors, or magnetic particles than can be attached to or incorporated into a polynucleotide, protein, or antibody. Visible labels include but are not limited to anthocyanins, fluorescein, green fluorescent protein (GFP), &bgr; glucuronidase, lissamine, luciferase, phycoerythrin, rhodamine, Cy3 and Cy5, and the like. Radioactive markers include radioactive forms of hydrogen, iodine, phosphorous, sulfur, and the like. “Ligand” refers to any agent, molecule, or compound which will bind specifically to a polynucleotide or to an epitope of a protein. Such ligands stabilize or modulate the activity of polynucleotides or proteins and may be composed of inorganic and/or organic substances including minerals, cofactors, nucleic acids, proteins, carbohydrates, fats, and lipids. “Oligonucleotide” refers a single-stranded molecule from about 18 to about 60 nucleotides in length which may be used in hybridization or amplification technologies or in regulation of replication, transcription or translation. Equivalent terms are amplicon, amplimer, primer, and oligomer. “Post-translational modification” of a protein can involve lipidation, glycosylation, phosphorylation, acetylation, racemization, proteolytic cleavage, and the like. These processes may occur synthetically or biochemically. Biochemical modifications will vary by cellular location, cell type, pH, enzymatic milieu, and the like. “Probe” refers to a cDNA that hybridizes to a nucleic acid or specifically binds to a ligand. Probes can be labeled with reporter molecules for use in hybridization technologies including Southern, northern, in situ, dot blot, and array, or in screening assays. “Protein” refers to a polypeptide or any portion thereof. A “portion” of a protein refers to that length of amino acid sequence which would retain at least one biological activity, a domain identified by PFAM or PRINTS analysis or an antigenic determinant of the protein identified using Kyte-Doolittle algorithms of the PROTEAN program (DNASTAR, Madison Wis.). An “oligopeptide” is an amino acid sequence from about five residues to about 15 residues that is used as part of a fusion protein to produce an antibody. “Sample” is used in its broadest sense as containing nucleic acids, proteins, and antibodies. A sample may comprise a bodily fluid such as ascites, blood, cerebrospinal fluid, lymph, semen, sputum, urine and the like; the soluble fraction of a cell preparation, or an aliquot of media in which cells were grown; a chromosome, an organelle, or membrane isolated or extracted from a cell; genomic DNA, RNA, or cDNA in solution or bound to a substrate; a cell; a tissue, a tissue biopsy, or a tissue print; buccal cells, skin, hair, a hair follicle; and the like. “Specific binding” refers to a precise interaction between two molecules which is dependent upon their structure, particularly their molecular side groups. For example, the intercalation of a regulatory protein into the major groove of a DNA molecule or the binding between an epitope of a protein and an agonist, antagonist, or antibody. “Substrate” refers to any rigid or semi-rigid support to which cDNAs, proteins, or antibodies are bound and includes membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, capillaries or other tubing, plates, polymers, and microparticles with a variety of surface forms including wells, trenches, pins, channels and pores.
[0035] A “transcript image” (TI) is a profile of gene transcription activity in a particular tissue at a particular time. TI provides assessment of the relative abundance of expressed polynucleotides in the cDNA libraries of an EST database as described in U.S. Pat. No. 5,840,484, incorporated herein by reference. “Variant” refers to molecules that are recognized variations of a protein or the polynucleotides that encode it. Splice variants may be determined by BLAST score, wherein the score is at least 100, and most preferably at least 400. Allelic variants have a high percent identity to the cDNAs and may differ by about three bases per hundred bases. “Single nucleotide polymorphism” (SNP) refers to a change in a single base as a result of a substitution, insertion or deletion. The change may be conservative (purine for purine) or non-conservative (purine to pyrimidine) and may or may not result in a change in an encoded amino acid or its secondary, tertiary, or quaternary structure. The Invention The present invention provides for a combination comprising a plurality of cDNAs or their complements, SEQ ID NOs: 1-501, which are differentially expressed in human prostate cancer cells treated with various growth factors and growth regulators relative to the untreated cells and to normal human prostate epithelial cells similarily treated, and which may be used to diagnose, to stage, to treat or to monitor the progression or treatment of prostate cancer. The composition may be used in its entirety or in part, as subsets of downregulated cDNAs, SEQ ID NOs: 1-56, 87-153, 164-349, 370-414, and 437-501, or of upregulated cDNAs, SEQ ID NOs:57-86, 154-163, 350-369, and 415-436.
[0036] SEQ ID NOs: 14, 26, 40, 52, 55, 60, 65, 68, 73, 79, 82, 85, 92, 110, 112, 114, 115, 117, 122, 125, 126, 130, 136, 137, 139, 141, 143, 144, 145, 146, 147, 160, 164, 166, 167, 168, 190, 191, 194, 195, 199, 201, 204, 211, 212, 222, 224, 226, 229, 233, 234, 240, 243, 245, 248, 250, 253, 254, 259, 264, 268, 269, 270, 272, 276, 278, 279, 281, 282, 284, 285, 286, 293, 296, 297, 299, 300, 301, 302, 306, 308, 312, 313, 314, 317, 319, 321, 321, 322, 322, 323, 324, 325, 326, 330, 331, 332, 334, 336, 337, 338, 339, 340, 342, 346, 353, 355, 357, 361, 365, 366, 371, 372, 376, 380, 383, 385, 386, 387, 390, 399, 400, 402, 405, 406, 408, 409, 410, 412, 413, 417, 418, 419, 420, 422, 422, 424, 426, 438, 444, 445, 453, 456, 460, 461, 471, 479, 480, 487, 490, 492, 495, 496, and 497 represent novel cDNAs differentially expressed in prostate cancer cells. Since the novel cDNAs were identified solely by their differential expression, it is not essential to know a priori the name, structure, or function of the gene or it's encoded protein. The usefulness of the novel cDNAs exists in their immediate value as diagnostics for prostate cancer.
[0037] Table 1 shows those cDNAs having lower expression (two-fold or greater decrease) or higher expression (two-fold or greater increase) in prostate cancer cells following various growth factor and growth regulator treatments. Table 2 shows Pfam annotations of the cDNAs of the present invention. Columns 1 and 2 show the SEQ ID NO and TEMPLATE ID, respectively. Columns 3, 4, and 5 show the first nucleotide (START), last nucleotide (STOP), and reading frame, respectively, for the protein encoded by the cDNA and identified by Pfam analysis. Columns 6 and 7 show the Pfam description and E-values, respectively, corresponding to the protein domain encoded by the cDNA. Table 3 shows signal peptide and transmembrane regions predicted within the proteins encoded by the cDNAs of the present invention. Columns 1 and 2 show the SEQ ID NO and TEMPLATE ID, respectively. Columns 3, 4, and 5 show the first nucleotide (START), last nucleotide (STOP), and reading frame, respectively, for a protein encoded by the cDNA, and column 6 identifies the signal peptide (SP) or transmembrane (TM) domain of the protein. Table 4 shows the region of each cDNA encompassed by the clone present on a microarray and identified as differentially expressed. Columns 1 and 2 show the SEQ ID NO and TEMPLATE ID, respectively. Column 3 shows the CLONE ID and columns 4 and 5 show the first nucleotide (START) and last nucleotide (STOP) encompassed by the clone on the template.
[0038] The differential expression of the cDNAs as shown using microarray analysis define an expression profile for prostate cancer. Experimentally, differential expression of the cDNAs can be evaluated by methods including, but not limited to, differential display by spatial immobilization or by gel electrophoresis, genome mismatch scanning, representational discriminant analysis, clustering, transcript imaging and array technologies. These methods may be used alone or in combination.
[0039] The combination may be arranged on a substrate and hybridized with tissues from prostate cancer patients to identify which of the cDNAs are differentially expressed. If the patient has a known stage of prostate cancer, i.e., metastasis to brain, bone, or lung, this allows identification of those sequences of highest potential therapeutic value. In one embodiment, an additional set of cDNAs, such as cDNAs encoding signaling molecules, are arranged on the substrate with the combination. Such combinations may be useful in the elucidation of pathways which are affected in prostate cancer or to identify new, coexpressed, candidate, therapeutic molecules.
[0040] In another embodiment, the combination can be used for large scale genetic or gene expression analysis of a large number of novel, nucleic acid molecules. These samples are prepared by methods well known in the art and are from mammalian cells or tissues which are in a certain stage of development; have been treated with a known molecule or compound, such as a cytokine, growth factor, a drug, and the like; or have been extracted or biopsied from a mammal with a known or unknown condition, disorder, or disease before or after treatment. The sample nucleic acid molecules are hybridized to the combination for the purpose of defining a novel gene profile associated with that developmental stage, treatment, or disorder.
[0041] cDNAs and Their Use
[0042] cDNAs can be prepared by a variety of synthetic or enzymatic methods well known in the art. cDNAs can be synthesized, in whole or in part, using chemical methods well known in the art (Caruthers et al. (1980) Nucleic Acids Symp Ser (7) 215-233). Alternatively, cDNAs can be produced enzymatically or recombinantly, by in vitro or in vivo transcription.
[0043] Nucleotide analogs can be incorporated into cDNAs by methods well known in the art. The only requirement is that the incorporated analog must base pair with native purines or pyrimidines. For example, 2,6-diaminopurine can substitute for adenine and form stronger bonds with thymidine than those between adenine and thymidine. A weaker pair is formed when hypoxanthine is substituted for guanine and base pairs with cytosine. Additionally, cDNAs can include nucleotides that have been derivatized chemically or enzymatically. cDNAs can be synthesized on a substrate. Synthesis on the surface of a substrate may be accomplished using a chemical coupling procedure and a piezoelectric printing apparatus as described by Baldeschweiler et al. (PCT publication WO95/25 1116). Alternatively, the cDNAs can be synthesized on a substrate surface using a self-addressable electronic device that controls when reagents are added as described by Heller et al. (U.S. Pat. No. 5,605,662). cDNAs can be synthesized directly on a substrate by sequentially dispensing reagents for their synthesis on the substrate surface or by dispensing preformed DNA fragments to the substrate surface. Typical dispensers include a micropipette delivering solution to the substrate with a robotic system to control the position of the micropipette with respect to the substrate. There can be a multiplicity of dispensers so that reagents can be delivered to the reaction regions efficiently.
[0044] cDNAs can be immobilized on a substrate by covalent means such as by chemical bonding procedures or UV irradiation. In one method, a cDNA is bound to a glass surface which has been modified to contain epoxide or aldehyde groups. In another method, a cDNA is placed on a polylysine coated surface and UV cross-linked to it as described by Shalon et al. (WO95/35505). In yet another method, a cDNA is actively transported from a solution to a given position on a substrate by electrical means (Heller, supra). cDNAs do not have to be directly bound to the substrate, but rather can be bound to the substrate through a linker group. The linker groups are typically about 6 to 50 atoms long to provide exposure of the attached cDNA. Preferred linker groups include ethylene glycol oligomers, diamines, diacids and the like. Reactive groups on the substrate surface react with a terminal group of the linker to bind the linker to the substrate. The other terminus of the linker is then bound to the cDNA. Alternatively, polynucleotides, plasmids or cells can be arranged on a filter. In the latter case, cells are lysed, proteins and cellular components degraded, and the DNA is coupled to the filter by UV cross-linking.
[0045] The cDNAs may be used for a variety of purposes. For example, the combination of the invention may be used on an array. The array, in turn, can be used in high-throughput methods for detecting a related polynucleotide in a sample, screening a plurality of molecules or compounds to identify a ligand, diagnosing prostate cancer, or inhibiting or inactivating a therapeutically relevant gene related to the cDNA.
[0046] When the cDNAs of the invention are employed on an array, the cDNAs are arranged so that each cDNA is present at a specified location on the substrate. Because the cDNAs are at specified locations, the hybridization patterns and intensities, which together create a unique expression profile, can be interpreted in terms of expression levels of particular genes and can be correlated with a particular metabolic process, condition, disorder, disease, stage of disease, or treatment.
[0047] Hybridization
[0048] The cDNAs or fragments or complements thereof may be used in various hybridization technologies. The cDNAs may be labeled using a variety of reporter molecules by either PCR, recombinant, or enzymatic techniques. For example, a commercially available vector containing the cDNA is transcribed in the presence of an appropriate polymerase, such as 17 or SP6 polymerase, and at least one labeled nucleotide. Commercial kits are available for labeling and cleanup of such cDNAs. Radioactive (Amersham Biosciences (APB), Piscataway N.J.), fluorescent (Qiagen-Operon, Alameda Calif.), and chemiluminescent labeling (Promega, Madison Wis.) are well known in the art.
[0049] A cDNA may represent the complete coding region of an mRNA or be designed or derived from unique regions of the mRNA or genomic molecule, an intron, a 3′ untranslated region, or from a conserved motif. The cDNA is at least 18 contiguous nucleotides in length and is usually single stranded. Such a cDNA may be used under hybridization conditions that allow binding only to an identical sequence, a naturally occurring molecule encoding the same protein, or an allelic variant. Discovery of related human and mammalian sequences may also be accomplished using a pool of degenerate cDNAs and appropriate hybridization conditions. Generally, a cDNA for use in Southern or northern hybridizations may be from about 400 to about 6000 nucleotides long. Such cDNAs have high binding specificity in solution-based or substrate-based hybridizations. An oligonucleotide may be used to detect or quantify expression of a polynucleotide in a sample using PCR.
[0050] The stringency of hybridization is determined by G+C content of the cDNA, salt concentration, and temperature. In particular, stringency is increased by reducing the concentration of salt or raising the hybridization temperature. In solutions used for some membrane based hybridizations, addition of an organic solvent such as formamide allows the reaction to occur at a lower temperature. Hybridization may be performed with buffers, such as 5×saline sodium citrate (SSC) with 1% sodium dodecyl sulfate (SDS) at 60° C., that permit the formation of a hybridization complex between nucleic acid sequences that contain some mismatches. Subsequent washes are performed with buffers such as 0.2×SSC with 0.1% SDS at either 45° C. (medium stringency) or 650-68° C. (high stringency). At high stringency, hybridization complexes will remain stable only where the nucleic acids are completely complementary. In some membrane-based hybridizations, preferably 35% or most preferably 50%, formamide may be added to the hybridization solution to reduce the temperature at which hybridization is performed. Background signals may be reduced by the use of detergents such as Sarkosyl or TRITON X-100 (Sigma-Aldrich, St. Louis Mo.) and a blocking agent such as denatured salmon sperm DNA. Selection of components and conditions for hybridization are well known to those skilled in the art and are reviewed in Ausubel et al. (1997, Short Protocols in Molecular Biology, John Wiley & Sons, New York N.Y., Units 2.8-2.11, 3.18-3.19 and 4-6-4.9).
[0051] Dot-blot, slot-blot, low density and high density arrays are prepared and analyzed using methods known in the art. cDNAs from about 18 consecutive nucleotides to about 5000 consecutive nucleotides in length are contemplated by the invention and used in array technologies. Depending on the technology employed, the number of cDNAs on a substrate ranges from at least two to about 100,000. The high density array may be used to monitor the expression level of large numbers of genes simultaneously and to identify genetic variants, mutations, and SNPs. Such information may be used to determine gene function; to understand the genetic basis of a disorder; to diagnose a disorder; and to develop and monitor the activities of therapeutic agents being used to control or cure a disorder. (See, e.g., U.S. Pat. No. 5,474,796; WO95/11995; WO95/35505; U.S. Pat. No. 5,605,662; and U.S. Pat. No. 5,958,342.)
[0052] Screening and Purification Assays
[0053] A cDNA may be used to screen a library or a plurality of molecules or compounds for a ligand which specifically binds the cDNA. Ligands may be DNA molecules, RNA molecules, peptide nucleic acid molecules, peptides, proteins such as transcription factors, promoters, enhancers, repressors, and other proteins that regulate replication, transcription, or translation of the polynucleotide in the biological system. The assay involves combining the cDNA or a fragment thereof with the molecules or compounds under conditions that allow specific binding and detecting the bound cDNA to identify at least one ligand that specifically binds the cDNA.
[0054] In one embodiment, the cDNA may be incubated with a library of isolated and purified molecules or compounds and binding activity determined by methods such as a gel-retardation assay (U.S. Pat. No. 6,010,849) or a reticulocyte lysate transcriptional assay. In another embodiment, the cDNA may be incubated with nuclear extracts from biopsied and/or cultured cells and tissues. Specific binding between the cDNA and a molecule or compound in the nuclear extract is initially determined by gel shift assay. Protein binding may be confirmed by raising antibodies against the protein and adding the antibodies to the gel-retardation assay where specific binding will cause a supershift in the assay.
[0055] In another embodiment, the cDNA may be used to purify a ligand, molecule or compound using affinity chromatography methods well known in the art. In one embodiment, the cDNA is chemically reacted with cyanogen bromide groups on a polymeric resin or gel. Then a sample is passed over and reacts with or binds to the cDNA. The molecule or compound which is bound to the cDNA may be released from the cDNA by increasing the salt concentration of the flow-through medium and collected.
[0056] Protein Production and Uses
[0057] The full length cDNAs or fragment thereof may be used to produce purified proteins using recombinant DNA technologies described herein and taught in Ausubel (sura; Units 16.1-16.62). One of the advantages of producing proteins by these procedures is the ability to obtain highly-enriched sources of the proteins thereby simplifying purification procedures.
[0058] The proteins may contain amino acid substitutions, deletions or insertions made on the basis of similarity in polarity, charge, solubility, hydrophobicity, and/or the amphipathic nature of the residues involved. Such substitutions may be conservative in nature when the substituted residue has structural or chemical properties similar to the original residue (e.g., replacement of leucine with isoleucine or valine) or they may be nonconservative when the replacement residue is radically different (e.g., a glycine replaced by a tryptophan). Computer programs included in LASERGENE software (DNASTAR, Madison Wis.) and algorithms included in RasMol software (University of Massachusetts, Amherst Mass.) may be used to help determine which and how many amino acid residues in a particular portion of the protein may be substituted, inserted, or deleted without abolishing biological or immunological activity.
[0059] Expression of Encoded Proteins
[0060] Expression of a particular cDNA may be accomplished by cloning the cDNA into a vector and transforming this vector into a host cell. The cloning vector used for the construction of cDNA libraries in the LIFESEQ databases (Incyte Genomics, Palo Alto Calif.) may also be used for expression. Such vectors usually contain a promoter and a polylinker useful for cloning, priming, and transcription. An exemplary vector may also contain the promoter for &bgr;-galactosidase, an amino-terminal methionine and the subsequent seven amino acid residues of &bgr;-galactosidase. The vector may be transformed into competent E. coli cells. Induction of the isolated bacterial strain with isopropylthiogalactoside using standard methods will produce a fusion protein that contains an N terminal methionine, the first seven residues of &bgr;-galactosidase, about 15 residues of linker, and the protein encoded by the cDNA.
[0061] The cDNA may be shuttled into other vectors known to be useful for expression of protein in specific hosts. Oligonucleotides containing cloning sites and fragments of DNA sufficient to hybridize to stretches at both ends of the cDNA may be chemically synthesized by standard methods. These primers may then be used to amplify the desired fragments by PCR. The fragments may be digested with appropriate restriction enzymes under standard conditions and isolated using gel electrophoresis. Alternatively, similar fragments are produced by digestion of the cDNA with appropriate restriction enzymes and filled in with chemically synthesized oligonucleotides. Fragments of the coding sequence from more than one gene may be ligated together and expressed.
[0062] Signal sequences that dictate secretion of soluble proteins are particularly desirable as component parts of a recombinant sequence. For example, a chimeric protein may be expressed that includes one or more additional purification-facilitating domains. Such domains include, but are not limited to, metal-chelating domains that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex, Seattle Wash.). The inclusion of a cleavable-linker sequence such as ENTEROKINASEMAX (Invitrogen, San Diego Calif.) between the protein and the purification domain may also be used to recover the protein.
[0063] Suitable host cells may include, but are not limited to, mammalian cells such as Chinese Hamster Ovary (CHO) and human 293 cells, insect cells such as Sf9 cells, plant cells such as Nicotiana tabacum, yeast cells such as Saccharomyces cerevisiae, and bacteria such as E. coli. For each of these cell systems, a useful vector may also include an origin of replication and one or two selectable markers to allow selection in bacteria as well as in a transformed eukaryotic host. Vectors for use in eukaryotic host cells may require the addition of 3′ poly(A) tail if the cDNA lacks poly(A).
[0064] Additionally, the vector may contain promoters or enhancers that increase gene expression. Many promoters are known and used in the art. Most promoters are host specific and exemplary promoters includes SV40 promoters for CHO cells; T7 promoters for bacterial hosts; viral promoters and enhancers for plant cells; and PGH promoters for yeast. Adenoviral vectors with the rous sarcoma virus enhancer or retroviral vectors with long terminal repeat promoters may be used to drive protein expression in mammalian cell lines. Once homogeneous cultures of recombinant cells are obtained, large quantities of secreted soluble protein may be recovered from the conditioned medium and analyzed using chromatographic methods well known in the art. An alternative method for the production of large amounts of secreted protein involves the transformation of mammalian embryos and the recovery of the recombinant protein from milk produced by transgenic cows, goats, sheep, and the like.
[0065] In addition to recombinant production, proteins or portions thereof may be produced manually, using solid-phase techniques (Stewart et al. (1969) Solid-Phase Peptide Synthesis, W H Freeman, San Francisco Calif.; Merrifield (1963) J Am Chem Soc 5:2149-2154), or using machines such as the 431A peptide synthesizer (Applied Biosystems (ABI), Foster City Calif.). Proteins produced by any of the above methods may be used as pharmaceutical compositions to treat disorders associated with null or inadequate expression of the genomic sequence.
[0066] Screening and Purification Assays
[0067] A protein or a portion thereof produced using a cDNA of the invention may be used to screen a library or a plurality of molecules or compounds for a ligand with specific binding affinity or to purify a molecule or compound from a sample. The protein or portion thereof employed in such screening may be free in solution, affixed to an abiotic or biotic substrate, or located intracellularly. For example, viable or fixed prokaryotic host cells that are stably transformed with recombinant nucleic acids that have expressed and positioned a protein on their cell surface can be used in screening assays. The cells are screened against a library or a plurality of ligands and the specificity of binding or formation of complexes between the expressed protein and the ligand may be measured. The ligands may be agonists, antagonists, antibodies, DNA molecules, enhancers, small drug molecules, immunoglobulins, inhibitors, mimetics, peptide nucleic acid molecules, peptides, pharmaceutical agents, proteins, and regulatory proteins, repressors, RNA molecules, ribozymes, and transcription factors or any other test molecule or compound that specifically binds the protein. An exemplary assay involves combining the mammalian protein or a portion thereof with the molecules or compounds under conditions that allow specific binding and detecting the bound protein to identify at least one ligand that specifically binds the protein.
[0068] This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding the protein specifically compete with a test compound capable of binding to the protein or oligopeptide or fragment thereof. One method for high throughput screening using very small assay volumes and very small amounts of test compound is described in U.S. Pat. No. 5,876,946. Molecules or compounds identified by screening may be used in a model system to evaluate their toxicity, diagnostic, or therapeutic potential.
[0069] The protein may be used to purify a ligand from a sample. A method for using a protein to purify a ligand would involve combining the protein or a portion thereof with a sample under conditions to allow specific binding, recovering the bound protein, and using an appropriate chaotropic agent to separate the protein from the purified ligand.
[0070] Production of Antibodies
[0071] A protein encoded by a cDNA of the invention may be used to produce specific antibodies. Antibodies may be produced using an oligopeptide or a portion of the protein with inherent immunological activity. Methods for producing antibodies include: 1) injecting an animal, usually goats, rabbits, or mice, with the protein, or an antigenically-effective portion or an oligopeptide thereof, to induce an immune response; 2) engineering hybridomas to produce monoclonal antibodies; 3) inducing in vivo production in the lymphocyte population; or 4) screening libraries of recombinant immunoglobulins. Recombinant immunoglobulins may be produced as taught in U.S. Pat. No. 4,816,567.
[0072] Antibodies produced using the proteins of the invention are useful for the diagnosis of prepathologic disorders as well as the diagnosis of chronic or acute diseases characterized by abnormalities in the expression, amount, or distribution of the protein. A variety of protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies specific for proteins are well known in the art. Immunoassays typically involve the formation of complexes between a protein and its specific binding molecule or compound and the measurement of complex formation. Immunoassays may employ a two-site, monoclonal-based assay that utilizes monoclonal antibodies reactive to two noninterfering epitopes on a specific protein or a competitive binding assay (Pound (1998) Immunochemical Protocols, Humana Press, Totowa N.J.).
[0073] Immunoassay procedures may be used to quantify expression of the protein in cell cultures, in subjects with a particular disorder or in model animal systems under various conditions. Increased or decreased production of proteins as monitored by immunoassay may contribute to knowledge of the cellular activities associated with developmental pathways, engineered conditions or diseases, or treatment efficacy. The quantity of a given protein in a given tissue may be determined by performing immunoassays on freeze-thawed detergent extracts of biological samples and comparing the slope of the binding curves to binding curves generated by purified protein.
[0074] Labeling of Molecules for Assay
[0075] A wide variety of reporter molecules and conjugation techniques are known by those skilled in the art and may be used in various cDNA, polynucleotide, protein, peptide or antibody assays. Synthesis of labeled molecules may be achieved using commercial kits for incorporation of a labeled nucleotide such as 32P-dCTP, Cy3-dCTP or Cy5-dCTP or amino acid such as 35S-methionine. Polynucleotides, cDNAs, proteins, or antibodies may be directly labeled with a reporter molecule by chemical conjugation to amines, thiols and other groups present in the molecules using reagents such as BIODIPY or FITC (Molecular Probes, Eugene Oreg.).
[0076] The proteins and antibodies may be labeled for purposes of assay by joining them, either covalently or noncovalently, with a reporter molecule that provides for a detectable signal. A wide variety of labels and conjugation techniques are known and have been reported in the scientific and patent literature including, but not limited to U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241.
[0077] Diagnostics
[0078] The cDNAs, or fragments thereof, may be used to detect and quantify differential gene expression; absence, presence, or excess expression of mRNAs; or to monitor mRNA levels during therapeutic intervention of prostate cancer. These cDNAs can also be utilized as markers of treatment efficacy against prostate cancer over a period ranging from several days to months. The diagnostic assay may use hybridization or amplification technology to compare gene expression in a biological sample from a patient to standard samples in order to detect differential expression. Qualitative or quantitative methods for this comparison are well known in the art.
[0079] For example, the cDNA may be labeled by standard methods and added to a biological sample from a patient under conditions for hybridization complex formation. After an incubation period, the sample is washed and the amount of label (or signal) associated with hybridization complexes is quantified and compared with a standard value. If the amount of label in the patient sample is significantly altered in comparison to the standard value, then the presence of the associated condition, disease or disorder is indicated.
[0080] In order to provide a basis for the diagnosis of a disorder associated with prostate cancer, a normal or standard expression profile is established. This may be accomplished by combining a biological sample taken from normal subjects, either animal or human, with a probe under conditions for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained using normal subjects with values from an experiment in which a known amount of a purified target sequence is used. Standard values obtained in this manner may be compared with values obtained from samples from patients who are symptomatic for a particular condition, disease, or disorder. Deviation from standard values toward those associated with a particular condition is used to diagnose that condition.
[0081] Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies and in clinical trial or to monitor the treatment of an individual patient. Once the presence of a condition is established and a treatment protocol is initiated, diagnostic assays may be repeated on a regular basis to determine if the level of expression in the patient begins to approximate that which is observed in a normal subject. The results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.
[0082] Gene Expression Profiles
[0083] A gene expression profile comprises a plurality of proteins or cDNAs and a plurality of detectable complexes, wherein each complex is formed by specific binding between the protein or cDNA and a ligand in a in a sample. The cDNAs of the invention are used as elements on an array to analyze gene expression profiles. In one embodiment, the array is used to monitor the progression of disease. Researchers can assess and catalog the differences in gene expression between healthy and diseased tissues or cells. By analyzing changes in patterns of gene expression, disease can be diagnosed at earlier stages before the patient is symptomatic. The invention can be used to formulate a prognosis and to design a treatment regimen. The invention can also be used to monitor the efficacy of treatment. For treatments with known side effects, the array is employed to improve the treatment regimen. A dosage is established that causes a change in genetic expression patterns indicative of successful treatment. Expression patterns associated with the onset of undesirable side effects are avoided. This approach may be more sensitive and rapid than waiting for the patient to show inadequate improvement, or to manifest side effects, before altering the course of treatment.
[0084] Two-dimensional polyacrylamide gel electrophoresis, mass spectrophotometry, western analysis, ELISA, RIA, fluorescent activated cell sorting (FACS), and protein or antibody arrays are used to produce protein expression profiles. Protocols for detecting and measuring protein expression using labeling moieties appropriate to the protocol are well known in the art.
[0085] Experimentally, expression profiles can also be evaluated by methods including, but not limited to, differential display by spatial immobilization or by gel electrophoresis, genome mismatch scanning, representational discriminant analysis, clustering, transcript imaging, and by protein or antibody arrays. Expression profiles produced by these methods may be used alone or in combination. The correspondence between mRNA and protein expression has been discussed by Zweiger (2001, Transducing the Genome. McGraw-Hill, San Francisco, Calif.) and Glavas et al. (2001; T cell activation upregulates cyclic nucleotide phosphodiesterases 8A1 and 7A3, Proc Natl Acad Sci 98:6319-6342) among others.
[0086] In another embodiment, animal models which mimic a human disease can be used to characterize expression profiles associated with a particular condition, disorder or disease; or treatment of the condition, disorder or disease. Novel treatment regimens may be tested in these animal models using arrays to establish and then follow expression profiles over time. In addition, arrays may be used with cell cultures or tissues removed from animal models to rapidly screen large numbers of candidate drug molecules, looking for ones that produce an expression profile similar to those of known therapeutic drugs, with the expectation that molecules with the same expression profile will likely have similar therapeutic effects. Thus, the invention provides the means to rapidly determine the molecular mode of action of a drug.
[0087] Assays Using Antibodies
[0088] Antibodies directed against epitopes on a protein encoded by a cDNA of the invention may be used in assays to quantify the amount of protein found in a particular human cell. Such assays include methods utilizing the antibody and a label to detect expression level under normal or disease conditions. The antibodies may be used with or without modification, and labeled by joining them, either covalently or noncovalently, with a labeling moiety. Various immunoassays for proteins (also mentioned above) typically involve the formation of complexes between the protein and its specific antibody and the measurement of such complexes.
[0089] Antibody Arrays
[0090] In an alternative to yeast two hybrid system analysis of proteins, an antibody array can be used to study protein-protein interactions and phosphorylation. A variety of protein ligands are immobilized on a membrane using methods well known in the art. The array is incubated in the presence of cell lysate until protein:antibody complexes are formed. Proteins of interest are identified by exposing the membrane to an antibody specific to the protein of interest. In the alternative, a protein of interest is labeled with digoxigenin (DIG) and exposed to the membrane; then the membrane is exposed to anti-DIG antibody which reveals where the protein of interest forms a complex. The identity of the proteins with which the protein of interest interacts is determined by the position of the protein of interest on the membrane.
[0091] Antibody arrays can also be used for high-throughput screening of recombinant antibodies. Bacteria containing antibody genes are robotically-picked and gridded at high density (up to 18,342 different double-spotted clones) on a filter. Up to 15 antigens at a time are used to screen for clones to identify those that express binding antibody fragments. These antibody arrays can also be used to identify proteins which are differentially expressed in samples (de Wildt et al. (2000) Nature Biotechnol 18:989-94).
[0092] Therapeutics
[0093] The cDNAs can be used in gene therapy. cDNAs can be delivered ex vivo to target cells, such as cells of bone marrow. Once stable integration and transcription and or translation are confirmed, the bone marrow may be reintroduced into the subject. Expression of the protein encoded by the cDNA may correct a disorder associated with mutation of a normal sequence, reduction or loss of an endogenous protein, or overepression of an endogenous or mutant protein. Alternatively, cDNAs may be delivered in vivo using vectors such as retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, and bacterial plasmids. Non-viral methods of gene delivery include cationic liposomes, polylysine conjugates, artificial viral envelopes, and direct injection of DNA (Anderson (1998) Nature 392:25-30; Dachs et al. (1997) Oncol Res 9:313-325; Chu et al. (1998) J Mol Med 76(3-4):184-192; Weiss et al. (1999) Cell Mol Life Sci 55(3):334-358; Agrawal (1996) Antisense Therapeutics, Humana Press, Totowa N.J.; and August et al. (1997) Gene Therapy (Advances in Pharmacology, Vol. 40), Academic Press, San Diego Calif.).
[0094] In addition, expression of a particular protein can be regulated through the specific binding of a fragment of a cDNA to a genomic sequence or an mRNA which encodes the protein or directs its transcription or translation. The cDNA can be modified or derivatized to any RNA-like or DNA-like material including peptide nucleic acids, branched nucleic acids, and the like. These sequences can be produced biologically by transforming an appropriate host cell with a vector containing the sequence of interest.
[0095] Molecules which regulate the activity of the cDNA or encoded protein are useful as therapeutics for treating prostate cancer. Such molecules include agonists which increase the expression or activity of the polynucleotide or encoded protein, respectively; or antagonists which decrease expression or activity of the polynucleotide or encoded protein, respectively. In one aspect, an antibody which specifically binds the protein may be used directly as an antagonist or indirectly as a delivery mechanism for bringing a pharmaceutical agent to cells or tissues which express the protein.
[0096] Additionally, any of the proteins, or their ligands, or complementary nucleic acid sequences may be administered as pharmaceutical compositions or in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents may act synergistically to affect the treatment or prevention of the conditions and disorders associated with an immune response. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects. Further, the therapeutic agents may be combined with pharmaceutically-acceptable carriers including excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Further details on techniques for formulation and administration used by doctors and pharmacists may be found in the latest edition of Remington's Pharmaceutical Sciences (Mack Publishing, Easton Pa.).
[0097] Model Systems
[0098] Animal models may be used as bioassays where they exhibit a phenotypic response similar to that of humans and where exposure conditions are relevant to human exposures. Mammals are the most common models, and most infectious agent, cancer, drug, and toxicity studies are performed on rodents such as rats or mice because of low cost, availability, lifespan, reproductive potential, and abundant reference literature. Inbred and outbred rodent strains provide a convenient model for investigation of the physiological consequences of underexpression or overexpression of genes of interest and for the development of methods for diagnosis and treatment of diseases. A mammal inbred to overexpress a particular gene (for example, secreted in milk) may also serve as a convenient source of the protein expressed by that gene.
[0099] Transgenic Animal Models
[0100] Transgenic rodents that overexpress or underexpress a gene of interest may be inbred and used to model human diseases or to test therapeutic or toxic agents. (See, e.g., U.S. Pat. No. 5,175,383 and U.S. Pat. No. 5,767,337.) In some cases, the introduced gene may be activated at a specific time in a specific tissue type during fetal or postnatal development. Expression of the transgene is monitored by analysis of phenotype, of tissue-specific mRNA expression, or of serum and tissue protein levels in transgenic animals before, during, and after challenge with experimental drug therapies.
[0101] Embryonic Stem Cells
[0102] Embryonic (ES) stem cells isolated from rodent embryos retain the potential to form embryonic tissues. When ES cells such as the mouse 129/SvJ cell line are placed in a blastocyst from the C57BLJ6 mouse strain, they resume normal development and contribute to tissues of the live-born animal. ES cells are preferred for use in the creation of experimental knockout and knockin animals. The method for this process is well known in the art and the steps are: the cDNA is introduced into a vector, the vector is transformed into ES cells, transformed cells are identified and microinjected into mouse cell blastocysts, blastocysts are surgically transferred to pseudopregnant dams. The resulting chimeric progeny are genotyped and bred to produce heterozygous or homozygous strains.
[0103] Knockout Analysis
[0104] In gene knockout analysis, a region of a gene is enzymatically modified to include a non-natural intervening sequence such as the neomycin phosphotransferase gene (neo; Capecchi (1989) Science 244:1288-1292). The modified gene is transformed into cultured ES cells and integrates into the endogenous genome by homologous recombination. The inserted sequence disrupts transcription and translation of the endogenous gene.
[0105] Knockin Analysis
[0106] ES cells can be used to create knockin humanized animals or transgenic animal models of human diseases. With knockin technology, a region of a human gene is injected into animal ES cells, and the human sequence integrates into the animal cell genome. Transgenic progeny or inbred lines are studied and treated with potential pharmaceutical agents to obtain information on the progression and treatment of the analogous human condition.
[0107] As described herein, the uses of the cDNAs, provided in the Sequence Listing of this application, and their encoded proteins are exemplary of known techniques and are not intended to reflect any limitation on their use in any technique that would be known to the person of average skill in the art. Furthermore, the cDNAs provided in this application may be used in molecular biology techniques that have not yet been developed, provided the new techniques rely on properties of nucleotide sequences that are currently known to the person of ordinary skill in the art, e.g., the triplet genetic code, specific base pair interactions, and the like. Likewise, reference to a method may include combining more than one method for obtaining or assembling full length cDNA sequences that will be known to those skilled in the art. It is also to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. The examples below are provided to illustrate the subject invention and are not included for the purpose of limiting the invention.
EXAMPLES[0108] I Construction of cDNA Libraries
[0109] RNA was purchased from Clontech Laboratories (Palo Alto Calif.) or isolated from various tissues. Some tissues were homogenized and lysed in guanidinium isothiocyanate, while others were homogenized and lysed in phenol or in a suitable mixture of denaturants, such as TRIZOL reagent (Invitrogen). The resulting lysates were centrifuged over CsCl cushions or extracted with chloroform. RNA was precipitated with either isopropanol or ethanol and sodium acetate, or by other routine methods.
[0110] Phenol extraction and precipitation of RNA were repeated as necessary to increase RNA purity. In most cases, RNA was treated with DNAse. For most libraries, poly(A) RNA was isolated using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex particles (Qiagen, Valencia Calif.), or an OLIGOTEX mRNA purification kit (Qiagen). Alternatively, poly(A) RNA was isolated directly from tissue lysates using other kits, including the POLY(A)PURE mRNA purification kit (Ambion, Austin Tex.).
[0111] In some cases, Stratagene (La Jolla Calif.) was provided with RNA and constructed the corresponding cDNA libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed with the UNIZAP vector system (Stratagene) or SUPERSCRIPT plasmid system (Invitrogen) using the recommended procedures or similar methods known in the art. (See Ausubel, supra, Units 5.1 through 6.6.) Reverse transcription was initiated using oligo d(T) or random primers. Synthetic oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA was digested with the appropriate restriction enzyme or enzymes. For most libraries, the cDNA was size-selected (300-1000 bp) using SEPHACRYL S1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (APB) or preparative agarose gel electrophoresis. cDNAs were ligated into compatible restriction enzyme sites of the polylinker of the pBLUESCRIPT plasmid (Stratagene), pSPORT1 plasmid (Invitrogen), or pINCY plasmid (Incyte Genomics). Recombinant plasmids were transformed into XL1-BLUE, XL1-BLUEMRF, or SOLR competent E. coli cells (Stratagene) or DH5a, DH10B, or ELECTROMAX DH10B competent E. coli cells (Invitrogen).
[0112] In some cases, libraries were superinfected with a 5×excess of the helper phage, M13K07, according to the method of Vieira et al. (1987, Methods Enzymol 153:3-11) and normalized or subtracted using a methodology adapted from Soares (1994, Proc Natl Acad Sci 91:9228-9232), Swaroop et al. (1991, Nucl Acids Res 19:1954), and Bonaldo et al. (1996, Genome Research 6:791-806). The modified Soares normalization procedure was utilized to reduce the repetitive cloning of highly expressed high abundance cDNAs while maintaining the overall sequence complexity of the library. Modification included significantly longer hybridization times which allowed for increased gene discovery rates by biasing the normalized libraries toward those infrequently expressed low-abundance cDNAs which are poorly represented in a standard transcript image (Soares supra).
[0113] II Isolation and Sequencing of cDNA Clones
[0114] Plasmids were recovered from host cells by in vivo excision using the UNIZAP vector system (Stratagene) or by cell lysis. Plasmids were purified using one of the following: the Magic or WIZARD MINIPREPS DNA purification system (Promega); the AGTC MINIPREP purification kit (Edge BioSystems, Gaithersburg Md.); the QIAWELL 8, QIAWELL 8 Plus, or QIAWELL 8 Ultra plasmid purification systems, or the REAL PREP 96 plasmid purification kit (Qiagen). Following precipitation, plasmids were resuspended in 0.1 ml of distilled water and stored, with or without lyophilization, at 4° C.
[0115] Alternatively, plasmid DNA was amplified from host cell lysates using direct link PCR in a high-throughput format (Rao (1994) Anal Biochem 216:1-14). Host cell lysis and thermal cycling steps were carried out in a single reaction mixture. Samples were processed and stored in 384-well plates, and the concentration of amplified plasmid DNA was quantified fluorometrically using PICOGREEN dye (Molecular Probes) and a FLUOROSKAN II fluorescence scanner (Labsystems Oy, Helsinki, Finland). cDNA sequencing reactions were processed using standard methods or high-throughput instrumentation such as the CATALYST 800 thermal cycler (ABI) or the DNA ENGINE thermal cycler (MJ Research, Watertown Mass.) in conjunction with the HYDRA microdispenser (Robbins Scientific, Sunnyvale Calif.) or the MICROLAB 2200 system (Hamilton, Reno Nev.). cDNA sequencing reactions were prepared using reagents provided by APB or supplied in ABI sequencing kits such as the PRISM BIGDYE cycle sequencing kit (ABI). Electrophoretic separation of cDNA sequencing reactions and detection of labeled cDNAs were carried out using the MEGABACE 1000 DNA sequencing system (APB); the PRISM 373 or 377 sequencing systems (ABI) in conjunction with standard ABI protocols and base calling software; or other sequence analysis systems known in the art. Reading frames within the cDNA sequences were identified using standard methods (reviewed in Ausubel, supra, Unit 7.7).
[0116] III Extension of cDNA Sequences
[0117] Nucleic acid sequences were extended using the cDNA clones and oligonucleotide primers. One primer was synthesized to initiate 5′ extension of the known fragment, and the other, to initiate 3′ extension of the known fragment. The initial primers were designed using OLIGO software (Molecular Insights, Cascade Colo.), or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68° C. to about 72° C. Any stretch of nucleotides which would result in hairpin structures and primer-primer dimerizations was avoided.
[0118] Selected human cDNA libraries were used to extend the sequence. If more than one extension was necessary or desired, additional or nested sets of primers were designed. Preferred libraries are ones that have been size-selected to include larger cDNAs. Also, random primed libraries are preferred because they will contain more sequences with the 5′ and upstream regions of genes. A randomly primed library is particularly useful if an oligo d(T) library does not yield a full-length cDNA.
[0119] High fidelity amplification was obtained by PCR using methods well known in the art. PCR was performed in 96-well plates using the DNA ENGINE thermal cycler (MJ Research). The reaction mix contained DNA template, 200 mmol of each primer, reaction buffer containing Mg2+, (NH4)2SO4, and &bgr;-mercaptoethanol, Taq DNA polymerase (APB), ELONGASE enzyme (Invitrogen), and Pfu DNA polymerase (Stratagene), with the following parameters for primer pair PCI A and PCI B (Incyte Genomics): 1: 94° C., 3 min; 2: 94° C., 15 sec; 3: 60° C., 1 min; 4: 68° C., 2 min; 5: 2, 3, and 4 repeated 20 times; 6: 68° C., 5 min; and 7: storage at 4° C. In the alternative, the parameters for primer pair T7 and SK+(Stratagene) were as follows: 1: 94° C., 3 min; 2: 94° C., 15 sec; 3: 57° C., 1 min; 4: 68° C., 2 min; 5: 2, 3, and 4 repeated 20 times; 6: 68° C., 5 min; and 7: storage at 4° C.
[0120] The concentration of DNA in each well was determined by dispensing 100 &mgr;l PICOGREEN reagent (0.25% reagent in 1×TE, v/v; Molecular Probes) and 0.5 &mgr;l of undiluted PCR product into each well of an opaque fluorimeter plate (Corning Costar, Acton Mass.) and allowing the DNA to bind to the reagent. The plate was scanned in a FLUOROSKAN II (Labsystems Oy) to measure the fluorescence of the sample and to quantify the concentration of DNA. A 5 &mgr;l to 10 &mgr;l aliquot of the reaction mixture was analyzed by electrophoresis on a 1% agarose mini-gel to determine which reactions were successful in extending the sequence.
[0121] The extended nucleic acids were desalted and concentrated, transferred to 384-well plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison Wis.), and sonicated or sheared prior to religation into pUC 18 vector (APB). For shotgun sequencing, the digested nucleic acids were separated on low concentration (0.6 to 0.8%) agarose gels, fragments were excised, and agar digested with AGARACE enzyme (Promega). Extended clones were religated using T4 DNA ligase (New England Biolabs, Beverly Mass.) into pUC18 vector (APB), treated with Pfu DNA polymerase (Stratagene) to fill-in restriction site overhangs, and transformed into competent E. coli cells. Transformed cells were selected on antibiotic-containing media, and individual colonies were picked and cultured overnight at 37° C. in 384-well plates in LB/2×carbenicillin liquid media.
[0122] The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerase (APB) and Pfu DNA polymerase (Stratagene) with the following parameters: 1: 94° C., 3 min; 2: 94° C., 15 sec; 3: 60° C., 1 min; 4: 72° C., 2 min; 5: s 2, 3, and 4 repeated 29 times; 6: 72° C., 5 min; and 7: storage at 4° C. DNA was quantified using PICOGREEN reagent (Molecular Probes) as described above. Samples with low DNA recoveries were reamplified using the same conditions described above. Samples were diluted with 20% dimethylsulfoxide (DMSO; 1:2, v/v), and sequenced using DYENAMIC energy transfer sequencing primers and the DYENAMIC DIRECT cycle sequencing kit (APB) or the PRISM BIGDYE terminator cycle sequencing kit (ABI).
[0123] IV Assembly and Analysis of Sequences
[0124] The nucleic acid sequences presented in the Sequence Listing may contain occasional sequencing errors and unidentified nucleotides (N) that reflect state-of-the-art technology at the time the cDNA was first sequenced. Occasional sequencing errors and Ns may be resolved and SNPs verified either by resequencing the cDNA or using algorithms to compare the alignment of multiple sequences covering the region in which the N or potential SNP occurs. The sequences used in the verification may be identified from any available database using BLAST analysis and aligned using a variety of alignment algorithms described in Ausubel (supra, unit 7.7) and in Meyers (1995; Molecular Biology and Biotechnology, Wiley VCH, New York N.Y., pp. 856-853).
[0125] Component nucleotide sequences from chromatograms were subjected to PHRED analysis (Phil Green, University of Wash., Seattle Wash.) and assigned a quality score. The sequences having at least a required quality score were subject to various pre-processing algorithms to eliminate low quality 3′ ends, vector and linker sequences, polyA tails, Alu repeats, mitochondrial and ribosomal sequences, bacterial contamination sequences, and sequences smaller than 50 base pairs. Sequences were screened using the BLOCK 2 program (Incyte Genomics), a motif analysis program based on sequence information contained in the SWISS-PROT and PROSITE databases (Bairoch et al. (1997) Nucleic Acids Res 25:217-221; Attwood et al. (1997) J Chem Inf Comput Sci 37:417-424).
[0126] Processed sequences were subjected to assembly procedures in which the sequences were assigned to bins, one sequence per bin. Sequences in each bin were assembled to produce consensus sequences, templates. Subsequent new sequences were added to existing bins using BLAST (Altschul (supra); Altschul et al. (supra); Karlin et al. (1988) Proc Natl Acad Sci 85:841-845), BLASTn (vers.1.4, WashU), and CROSSMATCH software (Phil Green, supra). Candidate pairs were identified as all BLAST hits having a quality score greater than or equal to 150. Alignments of at least 82% local identity were accepted into the bin. The component sequences from each bin were assembled using PHRAP (Phil Green, supra). Bins with several overlapping component sequences were assembled using DEEP PHRAP (Phil Green, supra).
[0127] Bins were compared against each other, and those having local similarity of at least 82% were combined and reassembled. Reassembled bins having templates of insufficient overlap (less than 95% local identity) were re-split. Assembled templates were also subjected to analysis by STITCHER/EXON MAPPER algorithms which analyzed the probabilities of the presence of splice variants, alternatively spliced exons, splice junctions, differential expression of alternative spliced genes across tissue types, disease states, and the like. These resulting bins were subjected to several rounds of the above assembly procedures to generate the template sequences found in the LIFESEQ GOLD database (Incyte Genomics).
[0128] The assembled templates were annotated using the following procedure. Template sequences were analyzed using BLASTn (vers. 2.0, NCBI) versus GBpri (GenBank vers. 116). “Hits” were defined as an exact match having from 95% local identity over 200 base pairs through 100% local identity over 100 base pairs, or a homolog match having an E-value equal to or greater than 1×10−8. (The “E-value” quantifies the statistical probability that a match between two sequences occurred by chance). The hits were subjected to frameshift FASTx versus GENPEPT (GenBank version 109). In this analysis, a homolog match was defined as having an E-value of 1×10−8. The assembly method used above was described in U.S. Ser. No. 09/276,534, filed Mar. 25, 1999, and the LIFESEQ GOLD user manual (Incyte Genomics).
[0129] Following assembly, template sequences were subjected to motif, BLAST, Hidden Markov Model (HMM; Pearson and Lipman (1988) Proc Natl Acad Sci 85:2444-2448; Smith and Waterman (1981) J Mol Biol 147:195-197), and functional analyses, and categorized in protein hierarchies using methods described in U.S. Ser. No. 08/812,290, filed Mar. 6, 1997; U.S. Ser. No. 08/947,845, filed Oct. 9, 1997; U.S. Pat. No. 5,953,727; and U.S. Ser. No. 09/034,807, filed Mar. 4, 1998. Template sequences may be further queried against public databases such as the GenBank rodent, mammalian, vertebrate, eukaryote, prokaryote, and human EST databases.
[0130] V Selection of Sequences, Microarray Preparation and Use
[0131] Incyte clones represent template sequences derived from the LIESEQ GOLD assembled human sequence database (Incyte Genomics). In cases where more than one clone was available for a particular template, the 5′-most clone in the template was used on the microarray. The HUMAN GENOME GEM series 1-3 microarrays (Incyte Genomics) contain 28,626 array elements which represent 10,068 annotated clusters and 18,558 unannotated clusters.
[0132] For the UNIGEM series microarrays (Incyte Genomics), Incyte clones were mapped to non-redundant Unigene clusters (Unigene database (build 46), NCBI; Shuler (1997) J Mol Med 75:694-698), and the 5′ clone with the strongest BLAST alignment (at least 90% identity and 100 bp overlap) was chosen, verified, and used in the construction of the microarray. The UNIGEM V microarray (Incyte Genomics) contains 7075 array elements which represent 4610 annotated genes and 2,184 unannotated clusters.
[0133] To construct microarrays, cDNAs were amplified from bacterial cells using primers complementary to vector sequences flanking the cDNA insert. Thirty cycles of PCR increased the initial quantity of cDNA from 1-2 ng to a final quantity greater than 5 &mgr;g. Amplified cDNAs were then purified using SEPHACRYL-400 columns (APB). Purified cDNAs were immobilized on polymer-coated glass slides. Glass microscope slides (Corning, Corning N.Y.) were cleaned by ultrasound in 0.1% SDS and acetone, with extensive distilled water washes between and after treatments. Glass slides were etched in 4% hydrofluoric acid (VWR Scientific Products, West Chester Pa.), washed extensively in distilled water, and coated with 0.05% aminopropyl silane (Sigma Aldrich, St Louis Mo.) in 95% ethanol. Coated slides were cured in a 110° C. oven. cDNAs were applied to the coated glass substrate using a procedure described in U.S. Pat. No. 5,807,522. One microliter of the cDNA at an average concentration of 100 ng/ul was loaded into the open capillary printing element by a high-speed robotic apparatus which then deposited about 5 nl of cDNA per slide.
[0134] Microarrays were UV-crosslinked using a STRATALINKER UV-crosslinker (Stratagene), and then washed at room temperature once in 0.2% SDS and three times in distilled water. Non-specific binding sites were blocked by incubation of microarrays in 0.2% casein in phosphate buffered saline (Tropix, Bedford Mass.) for 30 minutes at 60° C. followed by washes in 0.2% SDS and distilled water as before.
[0135] Twenty-two UNIGEMV arrays were used to evaluate differential expression across experimental and control samples.
[0136] VI Preparation of Samples
[0137] Cell Growth and Treatments
[0138] The following cell lines were obtained from ATCC (Manassus Va.) and cultured in media according to the manufacturer's protocols: PrEC is a primary prostate epithelial cell line isolated from a normal donor; PC-3 is a prostate adenocarcinoma cell line isolated from a 62 year-old male with grade IV prostate adenocarcinoma metastasized to the bone; DU-145 is a prostate carcinoma cell line isolated from a 69 year-old man with widespread metastatic disease. DU-145 was isolated from a brain metastasis and has no detectable hormone sensitivity; LNCaP is a prostate carcinoma cell line isolated from a lymph node biopsy of a 50 year-old male with metastatic prostate carcinoma. LNCaP cells are responsive to 5-alpha-dihydrotestosterone and express androgen receptors. All cell cultures were incubated in low serum media 48 hours prior to treatment.
[0139] Cells were subjected to the following treatments: R1881 Androgen; PrEC and LNCaP cells were treated with 5 nM R1881 Androgen (Methyltrienolone) for 8, 14, 24, and 38 hrs. EGF; PrEC, LNCap, DU-145, and PC-3 cells were treated with 50 ng/ml of EGF for 4, 8, 14, 24, and 38 hrs. FGF; PrEC, DU-145, and PC-3 cells were treated with 50 ng/ml of FGF for 4, 8, 14, 24, and 38 hrs. TGF-&agr;; PrEC and PC-3 cells were treated with 50 ng/ml of TGF-&agr; for 8, 14, 24, and 36 hrs. TGF-&bgr;; PrEC and DU-145 cells were treated with 5 ng/ml of TGF-&bgr; for 4, 8, 14, 24, and 38 hrs. Cells were harvested at each time point and prepared as described below.
[0140] Isolation and Labeling of Sample Polynucleotides
[0141] Cells were harvested and lysed in 1 ml of TRIZOL reagent (5×106 cells/ml; Invitrogen). The lysates were vortexed thoroughly and incubated at room temperature for 2-3 minutes and extracted with 0.5 ml chloroform. The extract was mixed, incubated at room temperature for 5 minutes, and centrifuged at 15,000 rpm for 15 minutes at 4° C. The aqueous layer was collected and an equal volume of isopropanol was added. Samples were mixed, incubated at room temperature for 10 minutes, and centrifuged at 15,000 rpm for 20 minutes at 4° C. The supernatant was removed, and the RNA pellet was washed with 1 ml of 70% ethanol, centrifuged at 15,000 rpm at 4° C., and resuspended in RNAse-free water. The concentration of the RNA was determined by measuring the optical density at 260 nm.
[0142] Poly(A) RNA was prepared using an OLIGOTEX mRNA kit (Qiagen) with the following modifications: OLIGOTEX beads were washed in tubes instead of on spin columns, resuspended in elution buffer, and then loaded onto spin columns to recover mRNA. To obtain maximum yield, the mRNA was eluted twice.
[0143] Each poly(A) RNA sample was reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/&mgr;l oligo-d(T) primer (21mer), 1×first strand buffer, 0.03 units/ul RNAse inhibitor, 500 uM dATP, 500 uM dGTP, 500 uM dTTP, 40 uM dCTP, and 40 uM either dCTP-Cy3 or dCTP-CyS (APB). The reverse transcription reaction was performed in a 25 ml volume containing 200 ng poly(A) RNA using the GEMBRIGHT kit (Incyte Genomics). Specific control poly(A) RNAs (YCFR06, YCFR45, YCFR67, YCFR85, YCFR43, YCFR22, YCFR23, YCFR25, YCFR44, YCFR26) were synthesized by in vitro transcription from non-coding yeast genomic DNA (W. Lei, unpublished). As quantitative controls, control mRNAs (YCFR06, YCFR45, YCFR67, and YCFR85) at 0.002 ng, 0.02 ng, 0.2 ng, and 2 ng were diluted into reverse transcription reaction at ratios of 1:100,000, 1:10,000, 1:1000, 1:100 (w/w) to sample mRNA, respectively. To sample differential expression patterns, control mRNAs (YCFR43, YCFR22, YCFR23, YCFR25, YCFR44, YCFR26) were diluted into reverse transcription reaction at ratios of 1:3, 3:1, 1:10, 10:1, 1:25, 25:1 (w/w) to sample mRNA. Reactions were incubated at 37° C. for 2 hr, treated with 2.5 ml of 0.5M sodium hydroxide, and incubated for 20 minutes at 85° C. to the stop the reaction and degrade the RNA.
[0144] cDNAs were purified using two successive CHROMA SPIN 30 gel filtration spin columns (Clontech). Cy3- and CyS-labeled reaction samples were combined as described below and ethanol precipitated using 1 ml of glycogen (1 mg/ml), 60 ml sodium acetate, and 300 ml of 100% ethanol. The cDNAs were then dried to completion using a SpeedVAC system (Savant Instruments, Holbrook N.Y.) and resuspended in 14 &mgr;l 5×SSC/0.2% SDS.
[0145] VII Hybridization and Detection
[0146] Hybridization reactions contained 9 &mgr;l of sample mixture containing 0.2 &mgr;g each of Cy3 and Cy5 labeled cDNA synthesis products in 5×SSC, 0.2% SDS hybridization buffer. The mixture was heated to 65° C. for 5 minutes and was aliquoted onto the microarray surface and covered with an 1.8 cm2 coverslip. The microarrays were transferred to a waterproof chamber having a cavity just slightly larger than a microscope slide. The chamber was kept at 100% humidity internally by the addition of 140 &mgr;l of 5×SSC in a corner of the chamber. The chamber containing the microarrays was incubated for about 6.5 hours at 60° C. The microarrays were washed for 10 min at 45° C. in low stringency wash buffer (1×SSC, 0.1% SDS), three times for 10 minutes each at 45° C. in high stringency wash buffer (0.1×SSC), and dried.
[0147] Reporter-labeled hybridization complexes were detected with a microscope equipped with an Innova 70 mixed gas 10 W laser (Coherent, Santa Clara Calif.) capable of generating spectral lines at 488 nm for excitation of Cy3 and at 632 nm for excitation of Cy5. The excitation laser light was focused on the microarray using a 20×microscope objective (Nikon, Melville N.Y.). The slide containing the microarray was placed on a computer-controlled X-Y stage on the microscope and raster-scanned past the objective. The 1.8 cm×1.8 cm microarray used in the present example was scanned with a resolution of 20 micrometers.
[0148] In two separate scans, the mixed gas multiline laser excited the two fluorophores sequentially. Emitted light was split, based on wavelength, into two photomultiplier tube detectors (PMT R1477; Hamamatsu Photonics Systems, Bridgewater N.J.) corresponding to the two fluorophores. Appropriate filters positioned between the microarray and the photomultiplier tubes were used to filter the signals. The emission maxima of the fluorophores used were 565 nm for Cy3 and 650 nm for Cy5. Each microarray was typically scanned twice, one scan per fluorophore using the appropriate filters at the laser source, although the apparatus was capable of recording the spectra from both fluorophores simultaneously.
[0149] The sensitivity of the scans was calibrated using the signal intensity generated by a cDNA control species. Samples of the calibrating cDNA were separately labeled with the two fluorophores and identical amounts of each were added to the hybridization mixture. A specific location on the microarray contained a complementary DNA sequence, allowing the intensity of the signal at that location to be correlated with a weight ratio of hybridizing species of 1:100,000.
[0150] The output of the photomultiplier tube was digitized using a 12-bit RTI-835H analog-to-digital (A/D) conversion board (Analog Devices, Norwood, Mass.) installed in an IBM-compatible PC computer. The digitized data were displayed as an image where the signal intensity was mapped using a linear 20-color transformation to a pseudocolor scale ranging from blue (low signal) to red (high signal). The data was also analyzed quantitatively. Where two different fluorophores were excited and measured simultaneously, the data were first corrected for optical crosstalk (due to overlapping emission spectra) between the fluorophores using each fluorophore's emission spectrum.
[0151] A grid was superimposed over the fluorescence signal image such that the signal from each spot was centered in each element of the grid. The fluorescence signal within each element was then integrated to obtain a numerical value corresponding to the average intensity of the signal. The software used for signal analysis was the GEMTOOLS gene expression analysis program (Incyte Genomics). Significance was defined as signal to background ratio exceeding 2×and area hybridization exceeding 40%.
[0152] VIII Data Analysis and Results
[0153] Array elements that exhibited at least a two-fold change in expression at one or more time points, a signal intensity over 250 units, a signal-to-background ratio of at least 2.5, and an element spot size of at least 40% were identified as differentially expressed using the GEMTOOLS program (Incyte Genomics). Each of the treatment groups for the prostate tumor cell lines, LNCaP, DU-145, and PC-3 were compared with the untreated cell line, and with normal PrEC cells similarly treated. The cDNAs that are differentially expressed are shown in Table 1 and are divided into treatment groups, and by up- or down-regulated expression. The cDNAs are identified by their SEQ ID NO and TEMPLATE ID, and by the description associated with at least a fragment of a polynucleotide found in GenBank. The descriptions were obtained using the sequences of the Sequence Listing and BLAST analysis.
[0154] IX Other Hybridization Technologies and Analyses
[0155] Other hybridization technologies utilize a variety of substrates such as nylon membranes, capillary tubes, etc. Arranging cDNAs on polymer coated slides is described in EXAMPLE V; sample cDNA preparation and hybridization and analysis using polymer coated slides is described in EXAMPLES VI and VII, respectively.
[0156] The cDNAs are applied to a membrane substrate by one of the following methods. A mixture of cDNAs is fractionated by gel electrophoresis and transferred to a nylon membrane by capillary transfer. Alternatively, the cDNAs are individually ligated to a vector and inserted into bacterial host cells to form a library. The cDNAs are then arranged on a substrate by one of the following methods. In the first method, bacterial cells containing individual clones are robotically picked and arranged on a nylon membrane. The membrane is placed on LB agar containing selective agent (carbenicillin, kanamycin, ampicillin, or chloramphenicol depending on the vector used) and incubated at 37° C. for 16 hr. The membrane is removed from the agar and consecutively placed colony side up in 10% SDS, denaturing solution (1.5 M NaCl, 0.5 M NaOH), neutralizing solution (1.5 M NaCl, 1 M Tris, pH 8.0), and twice in 2×SSC for 10 min each. The membrane is then UV irradiated in a STRATALINKER UV-crosslinker (Stratagene).
[0157] In the second method, cDNAs are amplified from bacterial vectors by thirty cycles of PCR using primers complementary to vector sequences flanking the insert. PCR amplification increases a starting concentration of 1-2 ng nucleic acid to a final quantity greater than 5 &mgr;g. Amplified nucleic acids from about 400 bp to about 5000 bp in length are purified using SEPHACRYL-400 beads (APB). Purified nucleic acids are arranged on a nylon membrane manually or using a dot/slot blotting manifold and suction device and are immobilized by denaturation, neutralization, and UV irradiation as described above.
[0158] Hybridization probes derived from cDNAs of the Sequence Listing are employed for screening cDNAs, mRNAs, or genomic DNA in membrane-based hybridizations. Probes are prepared by diluting the cDNAs to a concentration of 40-50 ng in 45 &mgr;l TE buffer, denaturing by heating to 100° C. for five min, and briefly centrifuging. The denatured cDNA is then added to a REDIPRIME tube (APB), gently mixed until blue color is evenly distributed, and briefly centrifuged. Five microliters of [32P]dCTP is added to the tube, and the contents are incubated at 37° C. for 10 min. The labeling reaction is stopped by adding 5,ul of 0.2M EDTA, and probe is purified from unincorporated nucleotides using a PROBEQUANT G-50 microcolumn (APB). The purified probe is heated to 100° C. for five min, snap cooled for two min on ice.
[0159] Membranes are pre-hybridized in hybridization solution containing 1% Sarkosyl and 1×high phosphate buffer (0.5 M NaCl, 0.1 M Na2HPO4, 5 mM EDTA, pH 7) at 55° C. for two hr. The probe, diluted in 15 ml fresh hybridization solution, is then added to the membrane. The membrane is hybridized with the probe at 55° C. for 16 hr. Following hybridization, the membrane is washed for 15 min at 25° C. in 1 mM Tris (pH 8.0), 1% Sarkosyl, and four times for 15 min each at 25° C. in 1 mM Tris (pH 8.0). To detect hybridization complexes, XOMAT-AR film (Eastman Kodak, Rochester N.Y.) is exposed to the membrane overnight at −70° C., developed, and examined.
[0160] X Further Characterization of Differentially Expressed cDNAs and Proteins
[0161] Clones were compared with the sequences in the LIFESEQ Gold 5.1 database (Incyte Genomics) using BLAST analysis, and an Incyte template and its variants were chosen for each clone. The template and variants were compared with the sequences in the GenBank database using BLAST analysis to acquire annotation. The nucleotide sequences were translated into amino acid sequence which was compared against the sequences in the GENPEPT and other protein databases using BLAST analysis to acquire annotation and other characterization such as domains and structural and functional motifs.
[0162] Percent sequence identity can also be determined electronically for two or more amino acid or nucleic acid sequences using the MEGALIGN program of LASERGENE software (DNASTAR). The percent similarity between two amino acid sequences is calculated by dividing the length of sequence A, minus the number of gap residues in sequence A, minus the number of gap residues in sequence B, into the sum of the residue matches between sequence A and sequence B, times one hundred. Gaps of low or of no homology between the two amino acid sequences are not included in determining percentage similarity.
[0163] Sequences with conserved protein motifs may be searched using the BLOCKS search program. This program analyses sequence information contained in the Swiss-Prot and PROSITE databases and is useful for determining the classification of uncharacterized proteins translated from genomic or cDNA sequences (Bairoch.(supra); Attwood (supra). PROSITE database is a useful source for identifying functional or structural domains that are not detected using motifs due to extreme sequence divergence. Using weight matrices, these domains are calibrated against the SWISS-PROT database to obtain a measure of the chance distribution of the matches.
[0164] The PRINTS database can be searched using the BLIMPS search program to obtain protein family “fingerprints”. The PRINTS database complements the PROSITE database by exploiting groups of conserved motifs within sequence alignments to build characteristic signatures of different protein families. For both BLOCKS and PRINTS analyses, the cutoff scores for local similarity were: >1300=strong, 1000-1300=suggestive; for global similarity were: p<exp-3; and for strength (degree of correlation) were: >1300=strong, 1000-1300=weak.
[0165] XI Expression of the Encoded Protein
[0166] Expression and purification of a protein encoded by a cDNA of the invention is achieved using bacterial or virus-based expression systems. For expression in bacteria, cDNA is subcloned into a vector containing an antibiotic resistance gene and an inducible promoter that directs high levels of cDNA transcription. Examples of such promoters include, but are not limited to, the trp-lac (tac) hybrid promoter and the T5 or T7 bacteriophage promoter in conjunction with the lac operator regulatory element. Recombinant vectors are transformed into bacterial hosts, such as BL21(DE3). Antibiotic resistant bacteria express the protein upon induction with IPTG. Expression in eukaryotic cells is achieved by infecting Spodoptera frugiperda (Sf9) insect cells with recombinant baculovirus, Autographica californica nuclear polyhedrosis virus. The polyhedrin gene of baculovirus is replaced with the cDNA by either homologous recombination or bacterial-mediated transposition involving transfer plasmid intermediates. Viral infectivity is maintained and the strong polyhedrin promoter drives high levels of transcription.
[0167] For ease of purification, the protein is synthesized as a fusion protein with glutathione-S-transferase (GST; APB) or a similar alternative such as FLAG. The fusion protein is purified on immobilized glutathione under conditions that maintain protein activity and antigenicity. After purification, the GST moiety is proteolytically cleaved from the protein with thrombin. A fusion protein with FLAG, an 8-amino acid peptide, is purified using commercially available monoclonal and polyclonal anti-FLAG antibodies (Eastman Kodak, Rochester N.Y.).
[0168] XII Production of Specific Antibodies
[0169] A denatured protein from a reverse phase HPLC separation is obtained in quantities up to 75 mg. This denatured protein is used to immunize mice or rabbits following standard protocols. About 100 Itg is used to immunize a mouse, while up to 1 mg is used to immunize a rabbit. The denatured protein is radioiodinated and incubated with murine B-cell hybridomas to screen for monoclonal antibodies. About 20 mg of protein is sufficient for labeling and screening several thousand clones.
[0170] In another approach, the amino acid sequence translated from a cDNA of the invention is analyzed using PROTEAN software (DNASTAR) to determine regions of high antigenicity, essentially antigenically-effective epitopes of the protein. The optimal sequences for immunization are usually at the C-terminus, the N-terminus, and those intervening, hydrophilic regions of the protein that are likely to be exposed to the external environment when the protein is in its natural conformation. Typically, oligopeptides about 15 residues in length are synthesized using an 431 Peptide synthesizer (ABI) using Fmoc-chemistry and then coupled to keyhole limpet hemocyanin (KLH; Sigma Aldrich) by reaction with M-maleimidobenzoyl-N-hydroxysuccinimide ester. If necessary, a cysteine may be introduced at the N-terminus of the peptide to permit coupling to KLH. Rabbits are immunized with the oligopeptide-KLH complex in complete Freund's adjuvant. The resulting antisera are tested for antipeptide activity by binding the peptide to plastic, blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting with radioiodinated goat anti-rabbit IgG.
[0171] Hybridomas are prepared and screened using standard techniques. Hybridomas of interest are detected by screening with radioiodinated protein to identify those fusions producing a monoclonal antibody specific for the protein. In a typical protocol, wells of 96 well plates (FAST, Becton-Dickinson, Palo Alto Calif.) are coated with affinity-purified, specific rabbit-anti-mouse (or suitable anti-species Ig) antibodies at 10 mg/ml. The coated wells are blocked with 1% BSA and washed and exposed to supernatants from hybridomas. After incubation, the wells are exposed to radiolabeled protein at 1 mg/ml. Clones producing antibodies bind a quantity of labeled protein that is detectable above background.
[0172] Such clones are expanded and subjected to 2 cycles of cloning at 1 cell/3 wells. Cloned hybridomas are injected into pristane-treated mice to produce ascites, and monoclonal antibody is purified from the ascitic fluid by affinity chromatography on protein A (APB). Monoclonal antibodies with affinities of at least 108 M−1, preferably 109 to 1010 M−1 or stronger, are made by procedures well known in the art.
[0173] XIII Purification of Naturally Occurring Protein Using Specific Antibodies
[0174] Naturally occurring or recombinant protein is substantially purified by immunoaffinity chromatography using antibodies specific for the protein. An immunoaffinity column is constructed by covalently coupling the antibody to CNBr-activated SEPHAROSE resin (APB). Media containing the protein is passed over the immunoaffinity column, and the column is washed using high ionic strength buffers in the presence of detergent to allow preferential absorbance of the protein. After coupling, the protein is eluted from the column using a buffer of pH 2-3 or a high concentration of urea or thiocyanate ion to disrupt antibody/protein binding, and the protein is collected.
[0175] XIV Screening Molecules for Specific Binding with the cDNA or Protein
[0176] The cDNA or fragments thereof and the protein or portions thereof are labeled with 32P-dCTP, Cy3-dCTP, Cy5-dCTP (APB), or BIODIPY or FITC (Molecular Probes), respectively. Candidate molecules or compounds previously arranged on a substrate are incubated in the presence of labeled nucleic or amino acid. After incubation under conditions for either a cDNA or a protein, the substrate is washed, and any position on the substrate retaining label, which indicates specific binding or complex formation, is assayed. The binding molecule is identified by its arrayed position on the substrate. Data obtained using different concentrations of the nucleic acid or protein are used to calculate affinity between the labeled nucleic acid or protein and the bound molecule. High throughput screening using very small assay volumes and very small amounts of test compound is fully described in U.S. Pat. No. 5,876,946, incorporated by reference herein.
[0177] All patents and publications mentioned in the specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the field of molecular biology or related fields are intended to be within the scope of the following claims. 1 TABLE 1 SEQ ID TEMPLATE GI Cell N0 ID Number E-Value Annotation Line Androgen treated, downregulated 1 441269.2 g2895090 0 Human RalBP1-interacting protein (POB1) mRNA, complete cds. LNCaP 2 3161.5 g6996441 0 Human CTL1 gene. LNCaP 3 977955.7 g438372 0 Human mRNA for protein kinase C mu. LNCaP 4 350754.2 g307503 0 Human transglutaminase E3 (TGASE3) mRNA, complete cds. LNCaP 5 235191.4 g2062372 0 Human cyclin-selective ubiquitin carrier protein mRNA, complete cds. LNCaP 6 1099593.13 g1702923 0 Human mRNA for p0071 protein. LNCaP 7 1099593.2 g1702923 0 Human mRNA for p0071 protein. LNCaP 8 003161.7c g6996441 0 Human CTL1 gene. LNCaP 9 415650.5 g190663 0 Human prostate-specific membrane antigen (PSM) mRNA, complete cds. LNCaP 10 204401.1 g2970122 0 Human prostate-specific membrane antigen (PSM) gene, complete cds. LNCaP 11 347915.14 g1503997 0 Human mRNA for KIAA0207 gene, complete cds. LNCaP 12 997395.1 g307513 0 Human transducin-like enhancer protein (TLE3) mRNA, complete cds. LNCaP 13 332783.1 g2795902 0 Human clone 23860 mRNA sequence. LNCaP 14 422289.1 Incyte Unique LNCaP 15 899410.5 g4165324 0 Human plasma membrane calcium ATPase isoform 1 (ATP2B1) gene, alternative LNCaP splice products, partial cds. 16 404155.2 g1255601 0 Human mRNA for cGMP-dependent protein kinase type I alpha, complete cds. LNCaP 17 412065.22 g1374791 0 Human selenium-binding protein (hSBP) mRNA, complete cds. LNCaP 18 412065.2 g1374791 7.00E−92 Human selenium-binding protein (hSBP) mRNA, complete cds. LNCaP 19 345860.21 g29709 0 Human mRNA for cathepsin H (EC 3.4.22.16). LNCaP 20 199101.1 g1296629 0 Human mRNA for UDP-GalNAc: polypeptide N-acetylgalactosaminyl transferase LNCaP (GalNAc-T3). 21 196606.8c g2924334 0 Human mRNA for exportin (tRNA). LNCaP 22 475146.3 g619876 0 Human mRNA for 3-hydroxy-3-methylglutaryl CoA synthase. LNCaP 23 474069.8 g7020654 0 unnamed protein product [Homo sapiens] LNCaP 24 255115.2 g5881245 0 Human UDP-glucuronosyltransferase 2B15 (UGT2B15) mRNA, UGT2B15-Y85 LNCaP allele, complete cds. 25 255115.4 g3287472 0 Human C19steroid specific UDP-glucuronosyltransferase mRNA, complete cds. LNCaP 26 216331.1 Incyte Unique LNCaP 27 482517.3 g6721135 0 Homo sapiens chromosome 14 clone CTD-2547F10, complete LNCaP 28 480653.1 g6841247 0 Human HSPC299 mRNA, partial cds. LNCaP 29 978047.1 g2852631 0 Human clone 23649 and 23755 unknown mRNA, partial cds. LNCaP 30 330977.1c g183784 0 Human androgen receptor (hAR) gene sequence. LNCaP 31 343502.9 g3779225 0 Human secreted cement gland protein XAG-2 homolog (hAG-2/R) mRNA, LNCaP complete cds. 32 903104.11c g181122 0 Human cleavage signal 1 protein mRNA, complete cds. LNCaP 33 898068.6 g6714698 0 Human mRNA for sugar transporter (SLC2A6 gene). LNCaP 34 903104.1 g181122 5.00E−55 Human cleavage signal 1 protein mRNA, complete cds. LNCaP 35 903104.8 g181122 0 Human cleavage signal 1 protein mRNA, complete cds. LNCaP 36 412065.21 g1374791 0 Human selenium-binding protein (hSBP) mRNA, complete cds. LNCaP 37 399067.1 g3157804 6.60E−257 neuronal leucine-rich repeat protein LNCaP 38 237709.11 g6172220 0 Human SPON2 mRNA for spondin 2, complete cds. LNCaP 39 237709.5 g6172220 0 Human SPON2 mRNA for spondin 2, complete cds. LNCaP 40 216437.4 Incyte Unique LNCaP 41 255824.52 g178350 0 Human aldolase A mRNA, complete cds. LNCaP 42 27798.1 g6453517 0 Human mRNA; cDNA DKFZp434M1317 (from clone DKFZp434M1317). LNCaP 43 60671.7 g3043675 0 Human mRNA for KIAA0576 protein, partial cds. LNCaP 44 376085.9 g6453582 0 Human mRNA; cDNA DKFZp434G1221 (from clone DKFZp434G1221). LNCaP 45 404467.1 g4826465 1.20E−56 dJ287G14.2 (PUTATIVE novel seven transmembrane domain protein) LNCaP 46 903508.12 g5262490 0 Human mRNA; cDNA DKFZp564D0462 (from clone DKFZp564D0462). LNCaP 47 334387.1 g632497 0 Human cleavage stimulation factor 77 kDa subunit mRNA, complete cds. LNCaP 48 290403.5 g6808173 0 Human mRNA; cDNA DKFZp564M1178 (from clone DKFZp564M1178); partial LNCaP 49 391406.5 g187701 0 Human MHC protein homologous to chicken B complex protein mRNA, complete LNCaP 50 391406.24 g187701 0 Human MHC protein homologous to chicken B complex protein mRNA, complete LNCaP 51 474588.21 g339700 0 Human polyadenylate binding protein (TIA-1) mRNA, complete cds. LNCaP 52 124541.1 Incyte Unique LNCaP 53 246862.9 g28937 0 Human mRNA for mitochondrial ATP synthase (F1-ATPase) alpha subunit. LNCaP 54 246862.17 g28937 0 Human mRNA for mitochondrial ATP synthase (F1-ATPase) alpha subunit. LNCaP 55 13040.1 Incyte Unique LNCaP 56 236480.3 g1556398 0 Human mRNA for FAN protein. LNCaP Androgen treated, upregulated 57 314831.5 g31920 0 Human GST1-Hs mRNA for GTP-binding protein. LNCaP 58 391940.1 g32451 0 Human pHS1-2 mRNA with ORF homologous to membrane receptor proteins. LNCaP 59 454958.13 g182482 0 Human fibroblast collagenase inhibitor mRNA, complete cds. LNCaP 60 1090481.2c g189152 2.00E−12 Human oligodendrocyte myelin glycoprotein (OMG) exons 1-2; LNCaP neurofibromatosis 1 (NF1) exons 28-49; ecotropic viral integration site 2B (EVI2B) exons 1-2; ecotropic viral integration site 2A (EVI2A) exons 1-2; 61 238203.11 g340236 0 Human vinculin mRNA, complete cds. LNCaP 62 994057.21 g531475 0 Human PPP1CB mRNA. LNCaP 63 346730.5 g2463627 0 Human putative monocarboxylate transporter (MCT) mRNA, complete cds. LNCaP 64 346730.2c g2463627 0 Human putative monocarboxylate transporter (MCT) mRNA, complete cds. LNCaP 65 458903.1 g34055 7.00E−14 Human K7 gene for simple epithelial cell keratin K7 (exon 4). LNCaP 66 482336.2 g34067 0 Human mRNA fragment for mesothelial type II keratin K7. LNCaP 67 482336.14 g34067 0 Human mRNA fragment for mesothelial type II keratin K7. LNCaP 68 66522.1 Incyte Unique LNCaP 69 300294.3 g1256819 0 Human signal recognition particle (SRP54) mRNA, complete cds. LNCaP 70 474372.8 g6807723 0 Human mRNA; cDNA DKFZp434J1114 (from clone DKFZp434J1114); partial LNCaP 71 475028.7 g575271 0 Human SPHAR gene for cyclin-related protein. LNCaP 72 481223.3 g2909359 0 Human mRNA for Sox10 protein. LNCaP 73 332499.1 g7022927 9.00E−38 unnamed protein product [Homo sapiens] LNCaP 74 234340.7 g4007417 0 Human Ets transcription factor PDEF (PDEF) mRNA, complete cds. LNCaP 75 234340.15c g6721497 0 Human PSE mRNA for prostate ets, complete cds. LNCaP 76 255778.11 g1373172 0 Human NADH: ubiquinone oxidoreductase subunit B13 (B13) mRNA, complete LNCaP 77 317586.1 g3265061 0 Human N-acetyltransferase-1 (NAT1) gene, NAT1*26B allele, complete cds. LNCaP 78 1093574.1c g3335147 0 Human short form transcription factor C-MAF (c-maf) mRNA, complete cds. LNCaP 79 355658.1 Incyte Unique LNCaP 80 201342.4 g5199315 0 Human non-ocogenic Rho GTPase-specific GTP exchange factor (proto-LBC) LNCaP mRNA, complete cds. 81 204542.1 g6425039 0 Human N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase LNCaP mRNA, complete cds. 82 428742.1 Incyte Unique LNCaP 83 204386.1 g7020228 1.00E−128 unnamed protein product [Homo sapiens] LNCaP 84 335086.1 g1543067 0 Human Has2 mRNA, complete cds. LNCaP 85 228302.1 Incyte Unique LNCaP 86 1063057.1 g488552 0 Human zinc finger protein ZNF134 mRNA, complete cds. LNCaP EGF treated, downregulated 87 199905.1 g3449309 0 Human mRNA for MEGF9, partial cds. DU145 88 11329.1 g4835609 0 Homo sapiens genomic DNA, chromosome 21q22.1, D21S226-AML LNCaP 89 237536.18 g4165090 0 Human NADH-ubiquinone oxidoreductase PDSW subunit mRNA, complete cds. DU145 90 330878.6 g5565654 0 Human cullin 4A (CUL4A) mRNA, complete cds. DU145 91 331793.11 g5262498 0 Human mRNA; cDNA DKFZp564G2362 (from clone DKFZp564G2362). DU145 92 902895.2 g4003380 5.00E−09 Human genomic DNA of 8p21.3-p22 anti-oncogene of hepatocellular colorectal DU145 and non-small cell lung cancer, segment 3/11. 93 7273.1 g5918013 0 Human DNA sequence from clone 423B22 on chromosome 1p33-35.3, PC3 94 190771.2 g5911950 0 Human mRNA; cDNA DKFZp727G051 (from clone DKFZp727G051); partial DU145 95 1095702.14 g187282 0 Human cation-dependent mannose 6-phosphate-specific receptor mRNA, DU145 complete cds. 96 1095702.4c g187282 0 Human cation-dependent mannose 6-phosphate-specific receptor mRNA, DU145 complete cds. 97 977667.1 g179699 0 Human C5a anaphylatoxin receptor mRNA, complete cds. DU145 98 253534.14 g5410450 0 Human interferon-induced protein p78 (MX1) gene, complete cds. PC3 99 1136709.5 g187273 0 Human eosinophil Charcot-Leyden crystal (CLC) protein (lysophospholipase) PC3 mRNA, complete cds. 100 1136709.6 g187273 0 Human eosinophil Charcot-Leyden crystal (CLC) protein (lysophospholipase) PC3 mRNA, complete cds. 101 382293.16 g3676496 0 Human inducible 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase (IPFK-2) DU145 mRNA, complete cds. 102 382293.15 g29542 0 Human mRNA for complement component Cls. DU145 103 235943.39c g37053 0 Human mRNA for transmembrane epithelial tumour mucin antigen. DU145 104 235943.36 g2055365 0 Human polymorphic epithelial mucin (PEM) gene, complete cds. DU145 105 401434.1 g4914611 0 Human mRNA; cDNA DKFZp586B2023 (from clone DKFZp586B2023). DU145 106 234107.2 g603559 0 Human LU gene for Lutheran blood group glycoprotein. DU145 107 233660.2c g3879501 1.10E−41 similar to ubiquitin carboxyl-terminal hydrolase; cDNA EST EMBL: D33366 DU145 comes from this gene; cDNA EST EMBL: D33965 comes from this gene; cDNA EST EMBL: D33822 comes from this gene; cDNA EST EMBL: D34547 comes from this gene; cDNA EST EMBL: D37684 108 482423.1 g2808656 0 Human complete genomic sequence between D16S3070 and D16S3275, DU145 containing Familial Mediterranean Fever gene disease. 109 441298.14 g3327137 0 Human mRNA for KIAA0662 protein, partial cds. DU145 110 235333.1 Incyte Unique DU145 111 221872.11c g5771534 0 Human secreted protein of unknown function (SPUF), mRNA, complete cds. DU145 112 992317.12 Incyte Unique DU145 113 230936.6 g4929618 0 Human CGI-75 protein mRNA, complete cds. DU145 114 029508.1c DU145 115 984900.1 g3881473 8.7 ZK1037.11 [Caenorhabditis elegans] DU145 116 405158.1 g6807861 0 Human mRNA; cDNA DKFZp434A0225 (from clone DKFZp434A0225). DU145 117 334177.1 Incyte Unique DU145 118 480187.81 g338928 0 Human T-cell receptor active beta-chain V-D-J-beta-1.2-C-beta-1 (TCRB) DU145 mRNA, partial cds. 119 62042.4 g6808161 0 Human mRNA; cDNA DKFZp761O051 (from clone DKFZp761O051). DU145 120 1087696.7 g4929632 0 Human CGI-82 protein mRNA, complete cds. DU145 121 259805.28 g189511 0 Human protein p78 mRNA, complete cds. DU145 122 399128.1 Incyte Unique DU145 123 986565.15 g5514676 0 Human mRNA for thiamine transporter (THTR-1), partial. DU145 124 978378.3 g4001636 5.00E−41 Human DNA, anonymous heat-stable fragment RP8-8. DU145 125 979575.2c DU145 126 233594.4 g6721505 0.85 hypothetical protein [Oryza sativa] DU145 127 238635.1 g219534 0 Human CGM1a mRNA for CD66d. DU145 128 1080496.1 g4500232 0 Human mRNA; cDNA DKFZp586B1122 (from clone DKFZp586B1122). DU145 129 351204.2 g35687 0 Human mRNA for protease 3. DU145 130 235394.5 Incyte Unique DU145 131 403242.1 g5725470 0 Human mRNA full length insert cDNA clone EUROIMAGE 1035904. DU145 132 427529.9 g4884083 0 Human mRNA; cDNA DKFZp564O243 (from clone DKFZp564O243); partial DU145 133 474868.2 g3603460 0 Human heat shock protein hsp40-3 mRNA, complete cds. DU145 134 1095839.17 g4929742 0 Human CGI-137 protein mRNA, complete cds. DU145 135 1095839.1 g4050043 0 Human RAD17 isoform 4 (RAD17) mRNA, complete cds. DU145 136 244251.4 Incyte Unique DU145 137 21555.1 Incyte Unique DU145 138 234157.3 g6716409 0 Human chromosome 16 open reading frame 5 (C16orf5) mRNA, complete cds. LNCaP 139 903478.1 Incyte Unique LNCaP 140 411296.2 g183911 0 Human hemopoietic cell protein-tyrosine kinase (HCK) gene, complete cds, clone DU145 lambda-a2/1a. 141 18044.1 g4929732 1.00E−15 Human CGI-132 protein mRNA, complete cds. DU145 142 407260.2 g7022708 0 unnamed protein product [Homo sapiens] PC3 143 978765.2 Incyte Unique DU145 144 338274.1 Incyte Unique DU145 145 231270.1 g829258 3.00E−04 Chitinase [Beta vulgaris] DU145 146 18092.1 Incyte Unique DU145 147 404197.8c DU145 148 404197.4 g3746548 0 Human cyclin K (CPR4) mRNA, complete cds. DU145 149 5460.1 g4914518 0 Human DNA sequence from clone CTA-216E10 on chromosome 22 DU145 150 346511.4 g338651 0 Human 69 kDa 2′5′ oligoadenylate synthetase (P69 2-5A synthetase) mRNA, PC3 complete cds. 151 346511.6 g338653 0 Human 71 kDa 2′5′ oligoadenylate synthetase (p69 2-5A synthetase) mRNA, PC3 complete cds. 152 331233.2 g437379 0 Human potassium voltage-gated channel (KCNC1) mRNA. DU145 153 234056.5 g6164748 0 Human F-box protein Fbx23 (FBX23) mRNA, partial cds. DU145 EGF treated, upregulated 154 238118.1 g4558635 0 Homo sapiens chromosome 19, BAC 82621 (CIT-B-139a18), complete PC3 155 233596.5 g7209311 2.00E−40 FLJ00005 protein [Homo sapiens] PC3 156 988653.1 g31129 0 Human mRNA for early growth response protein 1 (hEGR1). LNCaP 157 470468.26 g1050524 0 Human mRNA for uridine phosphorylase. DU145 158 470468.25 g1050524 0 Human mRNA for uridine phosphorylase. DU145 159 903479.3 g3882330 0 Human mRNA for KIAA0805 protein, partial cds. PC3 160 267918.1 Incyte Unique PC3 161 1094412.1 g1143491 0 Human mRNA for BiP protein. DU145 162 1094107.1 g1143491 0 Human mRNA for BiP protein. DU145 163 218452.4 g575265 0 Human PPP1R3 mRNA for protein phosphatase 1, glycogen-binding regulatory PC3 subunit. FGF treated, downregulated 164 12178.2 Incyte Unique DU145 165 100653.3 g436999 0 Human HRY gene, complete cds. PC3 166 978478.4 Incyte Unique PC3 167 85942.2 Incyte Unique PC3 168 85942.3 Incyte Unique PC3 169 977470.16c g392426 0 Human zinc-finger protein (bcl-6) mRNA, complete cds. PC3 & DU14 170 283762.2 g1469867 0 Human mRNA for KIAA0143 gene, partial cds. PC3 171 331065.2 g537293 0 Human negative regulator of programmed cell death ICH-1S (Ich-1) mRNA, PC3 complete cds. 172 331065.5 g537291 0 Human positive regulator of programmed cell death ICH-1L (Ich-1) mRNA, PC3 complete cds. 173 252782.4 g6103638 0 Human F-box protein FBL5 mRNA, partial cds. PC3 174 229068.1 g2687858 4.00E−62 renal organic anion transporter [Pseudopleuronectes americanus] DU145 175 407450.7 g1066391 0 Human t(3; 5)(q25.1; p34) fusion gene NPM-MLF1 mRNA, complete cds. DU145 176 200163.2 g5817262 0 Human mRNA; cDNA DKFZp566C224 (from clone DKFZp566C224). DU145 177 200163.12c g5817262 0 Human mRNA; cDNA DKFZp566C224 (from clone DKFZp566C224). DU145 178 1328237.4 g453578 0 Human mRNA for proto-oncogene protein, complete cds. PC3 179 333965.1 g1695872 0 Human ser-thr protein kinase PK428 mRNA, complete cds. PC3 180 1328236.5 g474898 0 Human cellular growth-regulating protein mRNA, complete cds. PC3 181 995534.3 g2290529 0 Human WD repeat protein HAN11 mRNA, complete cds. DU145 182 1098887.1 g1698719 0 Human zinc finger protein mRNA, complete cds. DU145 183 268733.1 g4753278 1.00E−132 Homo sapiens PAC clone RP5-85011 from 7q31.2-q32, complete PC3 & DU14 184 470023.2 g177830 0 Human alpha-1-antitrypsin gene (S variant), complete cds. DU145 185 989966.8 g3377596 0 Human full length insert cDNA YO54H04. DU145 186 233925.5 g773643 0 Human heterogeneous ribonucleoprotein A0 mRNA, complete cds. PC3 187 405145.5 g3252778 0 Human mRNA for 3′,5′-cyclic GMP phosphodiesterase, complete cds. DU145 188 261982.8 g2306765 0 Human zinc finger helicase (Znf-HX) mRNA, complete cds. DU145 188 261982.8 g2306765 0 Human zinc finger helicase (Znf-HX) mRNA, complete cds. PC3 189 246285.1 g339432 0 Human (clone CR-3) teratocarcinoma-derived growth factor 3 (TDGF3) mRNA, DU145 complete cds. 190 228511.1 Incyte Unique DU145 191 40290.1 g1854440 4.9 polyprotein [Turnip mosaic virus] DU145 192 399465.3 g1469194 0 Human mRNA for KIAA0136 gene, partial cds. DU145 193 898850.21 g187382 0 Human microtubule-associated protein 4 mRNA, complete cds. PC3 & DU14 194 201356.1 Incyte Unique PC3 & DU14 195 223285.1 g30413 1.00E−12 Human dinucleotide repeat polymorphism at the D21S65 locus. DU145 196 318000.4 g5817188 0 Human mRNA; cDNA DKFZp566L034 (from clone DKFZp566L034). DU145 197 977887.1 g1504017 0 Human mRNA for KIAA0218 gene, complete cds. PC3 198 1094199.1 g2584879 0 Human thyroid hormone receptor activator molecule (TRAM-1) mRNA, complete PC3 199 1094984.12 g2331249 7.00E−25 Human Amplified in Breast Cancer (AIB1) mRNA, complete cds. PC3 200 244785.3 g4454679 0 Human NADH-ubiquinone oxidoreductase subunit B14.5B homolog mRNA, PC3 complete cds. 201 242278.1c DU145 202 113621.5 g5817115 0 Human mRNA; cDNA DKFZp586J021 (from clone DKFZp586J021). DU145 203 401532.2 g1574997 0 Human canalicular multispecific organic anion transporter (cMOAT), gene, PC3 complete cds. 204 006985.1c PC3 205 351209.14 g556808 0 Human genes for acid sphingomyelinase ASM. PC3 & DU14 206 351209.16 g402620 0 Human mRNA for sphingomyelinase. PC3 & DU14 207 475819.14 g6523730 0 Human DNA sequence from clone RP3-351K20 on chromosome DU145 207 475819.14 g7159799 1.00E−153 dJ351K20.1.1 (novel C3HC4 type Zinc finger (RING finger) DU145 208 241384.3 g179400 0 Human beta-D-galactosidase mRNA, complete cds. PC3 209 3428.1 g6006046 0 Homo sapiens 2q35 BAC RPCI11-1064L18 (Roswell Park Cancer DU145 210 244200.1 g2292903 0 Human GalNAc-T1 gene, 3′UTR. DU145 211 412484.3 Incyte Unique PC3 212 412484.8 Incyte Unique PC3 213 402187.16 g3811348 0 Human cytosolic phospholipase A2 beta (cPLA2 beta) precursor RNA, complete DU145 sequence. 214 903091.31 g1255988 0 Human dystrobrevin-delta mRNA, complete cds. DU145 215 903091.33c g1255992 0 Human dystrobrevin-gamma mRNA, complete cds. PC3 216 903091.16 g1256012 0 Human dystrobrevin-beta mRNA, complete cds. DU145 217 228447.29c g338442 0 Human general beta-spectrin (SPTBN1) mRNA, complete cds. DU145 218 475473.1 g437000 0 Human microtubule-associated protein 1B (MAP1B) gene, complete cds. DU145 219 354430.4 g1665822 0 Human mRNA for KIAA0280 gene, partial cds. DU145 220 468221.12c g2895078 0 Human tumor protein D53 (TPD52L1) mRNA, partial cds. DU145 221 468221.13 g1469919 0 Human D53 (hD53) mRNA, complete cds. DU145 222 399187.1c g576780 5.00E−20 Human cyclin F mRNA, complete cds. DU145 223 903338.12 g181528 0 Human defensin 1 protein mRNA, complete cds. DU145 224 208529.1 Incyte Unique PC3 225 1040667.52 g3882166 0 Human mRNA for KIAA0723 protein, complete cds. DU145 226 147665.1 g4512267 5.00E−19 Human DNA for Ig heavy-chain variable region, complete sequence, 1 of 5. DU145 227 218524.4 g559053 0 Human interleukin 8 receptor beta (IL8RB) mRNA, complete cds. DU145 228 346673.1 g4218433 1.00E−47 Human chromosome 22 CpG island DNA, genomic Mse1 fragment, clone PC3 & DU14 22CGIB49B9, complete read. 229 58775.1 Incyte Unique DU145 230 197086.1 g1881565 0 Human cosmid g1572c101, complete sequence. DU145 231 348143.7 g1381163 0 Human huntingtin interacting protein (HIP2) mRNA, complete cds. DU145 232 239552.3c g28976 0 Human mRNA for azurocidin. DU145 233 233003.6 Incyte Unique PC3 234 233003.2 Incyte Unique PC3 235 217860.1 g4589637 0 Human mRNA for KIAA0997 protein, complete cds. DU145 236 216189.2 g1580723 0 Human mRNA for Tob, complete cds. PC3 & DU14 237 343692.15c g5733823 0 Human ubiquilin mRNA, complete cds. PC3 & DU14 238 382906.16 g180142 0 Human CD53 glycoprotein mRNA, complete cds. DU145 239 482325.15 g3043649 0 Human mRNA for KIAA0563 protein, complete cds. PC3 240 64851.1 Incyte Unique DU145 241 346016.5 g4033734 0 Human spliceosomal protein SAP 155 mRNA, complete cds. DU145 242 346016.6 g4033734 0 Human spliceosomal protein SAP 155 mRNA, complete cds. DU145 243 222222.1 Incyte Unique DU145 244 413835.5 g2257985 0 Human kruppel-related zinc finger protein hcKrox mRNA, complete cds. PC3 245 978273.4 g567834 2.00E−13 Human (clone HG52) Z-crystallin/quinone reductase (CRYZ) gene sequence. DU145 246 474673.1 g687592 0 Human p190-B (p190-B) mRNA, complete cds. PC3 247 480286.1 g510989 0 Human genes for histones H2B.1 and H2A. DU145 248 402640.1 Incyte Unique DU145 249 232218.1 g473629 1.00E−64 Human (clone 1NIB-138) normalized cDNA library sequence. PC3 250 981919.1c PC3 251 229369.1 g3169209 0 Homo sapiens BAC clone CTA-300E22 from 7q21-q31.1, complete PC3 252 253946.17 g186353 0 Human membrane glycoprotein gp130 mRNA, complete cds. PC3 & DU14 253 13550.1 Incyte Unique DU145 254 997704.1 g5926684 5.00E−13 Human genomic DNA, chromosome 3p21.3, clone: 301 to 308, anti-oncogene PC3 region, section 5/5. 255 385608.45 g2827202 0 Human general transcription factor 2-I (GTF2I) mRNA, alternatively spliced PC3 product, complete cds. 256 385608.25c g2687639 0 Human general transcription factor 2-I pseudogene 1 (GTF2IP1) mRNA. PC3 257 385608.3 g2415381 0 Human TFII-I protein (TFII-I) mRNA, complete cds. PC3 258 78434.1 g571295 0 Human CRFB4 gene, partial cds. DU145 259 243134.1 g5678818 1.00E−08 Human FRG1 (FRG1) gene, complete cds; 5S ribosomal RNA gene, complete DU145 sequence; TUB4q and TIG2 pseudogenes, complete sequence. 260 196959.4c g1617112 0 Human Na+-D-glucose cotransport regulator gene. PC3 261 82168.5 g6175872 0 Human toll-like receptor 4 (TLR4) gene, TLR4A allele, complete cds. PC3 262 4360.1 g7023028 0 Homo sapiens cDNA FLJ10785 fis, clone NT2RP4000457, weakly DU145 263 412661.2 g415818 0 Human mki67a mRNA (long type) for antigen of monoclonal antibody Ki-67. DU145 264 206310.2 Incyte Unique PC3 265 346599.14c g178848 0 Human apolipoprotein E mRNA, complete cds. DU145 266 385608.47 g2827202 0 Human general transcription factor 2-I (GTF2I) mRNA, alternatively spliced PC3 product, complete cds. 267 385608.2 g2827206 0 Human general transcription factor 2-I (GTF2I) mRNA, alternatively spliced PC3 product, complete cds. 268 335202.1 Incyte Unique PC3 269 978402.3 Incyte Unique DU145 270 232146.1 Incyte Unique PC3 271 25757.1 g5923890 0 Human cyclophilin-related protein (NKTR) gene, complete cds. DU145 272 206860.2 Incyte Unique PC3 273 239797.3 g2909843 0 Human prostate stem cell antigen (PSCA) mRNA, complete cds. PC3 274 441328.12 g4102966 0 Human pre-mRNA splicing SR protein rA4 mRNA, partial cds. PC3 275 113975.1 g4827314 0 Homo sapiens BAC clone RP11-365F8 from 7q31.1-q31.2, complete PC3 276 427554.6 Incyte Unique DU145 277 360130.31c g4099608 0 Human cell division control-related protein 2b (hcdcrel2b) mRNA, complete cds. DU145 278 82154.23 Incyte Unique PC3 279 82154.24 Incyte Unique PC3 280 1137293.16 g3341991 1.00E−49 Human histone macroH2A1.2 mRNA, complete cds. PC3 281 994468.3 Incyte Unique PC3 282 994532.1 Incyte Unique PC3 283 90710.1 g2342595 0 Human DNA sequence from cosmid U221F2 on chromosome X. PC3 & DU14 284 304359.1c PC3 & DU14 285 12402.1 Incyte Unique DU145 286 92743.1 Incyte Unique PC3 287 18513.1 g6706246 0 Human DNA sequence from clone RP3-393D12 on chromosome PC3 288 477387.7 g1036447 6.00E−72 Human CpG island DNA genomic Msel fragment, clone 96f6, forward read PC3 cpg96f6.ftla. 289 350440.14c g190096 0 Human plasma membrane calcium-pumping ATPase (PMCA4) mRNA, complete PC3 & DU14 290 350440.15 g179162 0 Human plasma membrane calcium ATPase (hPMCA4) mRNA, complete cds. PC3 & DU14 291 992455.56c g6048967 0 Human clone H14 unknown mRNA. DU145 292 997395.4 g3483689 0 Human full length insert cDNA clone ZD61F11. PC3 293 17821.1 Incyte Unique PC3 294 201436.4c g6093233 0 Human mRNA; cDNA DKFZp566G1424 (from clone DKFZp566G1424). PC3 295 201436.3 g6093233 0 Human mRNA; cDNA DKFZp566G1424 (from clone DKFZp566G1424). PC3 296 198947.1 Incyte Unique DU145 297 1135407.1c DU145 298 200046.1 g4156141 0 Homo sapiens BAC clone RP11-436H22 from 2, complete sequence. DU145 299 981489.1 Incyte Unique DU145 300 19362.1 Incyte Unique PC3 301 217116.1 Incyte Unique PC3 302 330530.1 Incyte Unique PC3 303 401213.1 g598644 1.00E−57 Human HepG2 partial cDNA, clone hmd2d12m5. DU145 304 400280.3 g4514553 0 Human mRNA forRod1, complete cds. PC3 305 17320.1 g2736289 0 Human hMed7 (MED7) mRNA, complete cds. DU145 306 406182.1c PC3 307 244603.1 g6065863 0 Human DNA sequence from clone 1018E9 on chromosome PC3 308 107405.1 Incyte Unique PC3 309 337024.4 g299702 0 75 kda infertility-related sperm protein [Human, testis, mRNA Partial, 2427 nt]. DU145 310 337024.3 g299702 0 75 kda infertility-related sperm protein [Human, testis, mRNA Partial, 2427 nt]. DU145 311 481848.1c g2865218 0 Human integrin binding protein Del-1 (Del1) mRNA, complete cds. PC3 & DU14 312 208723.1 Incyte Unique DU145 313 64612.1 Incyte Unique PC3 314 978402.1 Incyte Unique DU145 315 453369.8 g6642986 0 Human aminopeptidase PILS (APPILS) mRNA, complete cds. DU145 316 27240.1 g5649181 1.00E−155 Homo sapiens 3q26.2-27 BAC RPCI11-469J4 (Roswell Park Cancer DU145 317 401290.1c DU145 318 481847.1 g2865220 0 Human integrin binding protein Del-1, Z20 splice variant, (Del1) mRNA, partial PC3 & DU14 319 235636.1 g2865218 1.00E−19 Human integrin binding protein Del-1 (Del1) mRNA, complete cds. PC3 & DU14 320 994057.7 g37137 3.00E−79 Human mRNA for thrombospondin. DU145 321 405559.1 g897824 1.70E−17 AHNAK gene product DU145 322 16760.1 Incyte Unique DU145 323 198522.1 Incyte Unique PC3 324 19366.1 Incyte Unique DU145 325 002679.7c PC3 326 208276.1 Incyte Unique DU145 327 21552.1 g3688074 0 Homo sapiens chromosome 5, BAC clone 34j15 (LBNL H169), PC3 328 198087.1 g4160665 0 Human cDNA for NG,NG-dimethylarginine dimethylaminohydrolase, complete PC3 329 903269.4 g3661609 0 Human splicing factor Prp8 mRNA, complete cds. PC3 330 351166.1 Incyte Unique DU145 331 344713.3 g6472600 0.29 unconventional myosin heavy chain [Chara corallina] DU145 332 167920.1 g2636170 1.7 similar to antibiotic resistance protein [Bacillus subtilis] DU145 333 433569.1 g791003 0 Human ARSE gene, complete cds. PC3 334 406365.1 Incyte Unique PC3 335 348121.9 g1136417 0 Human mRNA for KIAA0179 gene, partial cds. PC3 336 363585.1c PC3 337 408116.1 Incyte Unique PC3 338 339737.1c DU145 339 978146.1 Incyte Unique PC3 340 186012.1 Incyte Unique DU145 341 405041.1 g3929221 2.40E−102 TRF1-interacting ankyrin-related ADP-ribose polymerase DU145 342 474266.2 g3929218 7.00E−28 Human TRF1-interacting ankyrin-related ADP-ribose polymerase mRNA, DU145 complete cds. 343 401530.2 g6731234 4.00E−75 Human myoferlin (MYOF) mRNA, complete cds. DU145 344 234729.11 g6330496 0 Human mRNA for KIAA1207 protein, partial cds. DU145 345 195199.1 g6329818 0 Human mRNA for KIAA1131 protein, partial cds. DU145 346 368731.1 Incyte Unique PC3 347 372313.6 g885977 0 Human organic anion transporting polypeptide (OATP) mRNA, complete cds. PC3 348 256871.2 g3861482 2.00E−86 Human chromosome 3, olfactory receptor pseudogene cluster 1, complete DU145 sequence, and myosin light chain kinase (MLCK) pseudogene, partial sequence. 349 256871.16c g896064 0 Human protein immuno-reactive with anti-PTH polyclonal antibodies mRNA, DU145 partial cds. FGF treated, upregulated 350 22485.15 g34349 0 Human LFA-3 mRNA for glycosylated surface protein. DU145 351 22485.1 g34346 0 Human mRNA for lymphocyte function associated antigen-3 (LFA-3). DU145 352 26410.1 g2381480 0 Human mRNA for epiregulin, complete cds. DU145 353 215720.1 Incyte Unique DU145 354 472165.22 g662993 0 Human mRNA encoding GPI-anchored protein p137. DU145 355 206580.1 Incyte Unique DU145 356 344775.3 g3822553 0 nuclear calmodulin-binding protein [Gallus gallus] DU145 357 334668.1 Incyte Unique DU145 358 416874.3 g340159 0 Human pro-urokinase mRNA, complete cds. DU145 359 427964.2 g31462 0 Human fra-1 mRNA. DU145 360 234537.3c g23896 0 Human placental cDNA coding for 5′nucleotidase (EC 3.1.3.5). DU145 361 31760.1 Incyte Unique DU145 362 336953.7 g1813423 0 Human mRNA for HCS, complete cds. DU145 363 444771.2 g31107 0 Human mRNA for elongation factor 2. DU145 364 449173.16 g189499 0 Human p62 mRNA, complete cds. DU145 365 1523.1 Incyte Unique DU145 366 239097.1 Incyte Unique DU145 367 252747.27 g6330689 0 Human mRNA for KIAA1228 protein, partial cds. DU145 368 482490.11 g6650213 8.00E−69 Human RAN binding protein 16 mRNA, complete cds. DU145 369 1000084.27 g3719220 0 Human vascular endothelial growth factor mRNA, complete cds. DU145 TGF&bgr; treated, downregulated 263 412661.2 g415818 0 Human mki67a mRNA (long type) for antigen of monoclonal antibody Ki-67. DU145 316 27240.1 g5649181 1.00E−155 Homo sapiens 3q26.2-27 BAC RPCI11-469J4 (Roswell Park Cancer DU145 370 205607.5 g987947 0 Human mRNA for phosphatidylinositol 3-kinase. DU145 371 1089426.1 g886947 0.54 orf3 [Saccharomyces cerevisiae] DU145 372 335186.2 g3929751 2.00E−11 Human SYBL1 gene. DU145 373 357276.8 g2232030 0 Human inositol polyphosphate 4-phosphatase type I-beta mRNA, complete cds. DU145 374 474724.5 g507212 0 Human serine kinase mRNA, complete cds. DU145 375 230889.3 g5817126 0 Human mRNA; cDNA DKFZp586P1622 (from clone DKFZp586P1622). DU145 376 903909.1 Incyte Unique DU145 377 229298.1 g4500162 0 Human mRNA; cDNA DKFZp586D0918 (from clone DKFZp586D0918). DU145 378 229298.2 g4500162 0 Human mRNA; cDNA DKFZp586D0918 (from clone DKFZp586D0918). DU145 379 110678.1 g4371263 0 Homo sapiens chromosome 16 clone 66H6, complete sequence. DU145 380 239093.1 Incyte Unique DU145 381 237963.11c g3873560 0 Human mRNA for C17orf1 protein. DU145 382 237963.8 g3873560 0 Human mRNA for C17orf1 protein. DU145 383 400135.1 Incyte Unique DU145 384 344398.2 g4500170 0 Human mRNA; cDNA DKFZp586K1318 (from clone DKFZp586K1318). DU145 385 1086647.1 Incyte Unique DU145 386 1682.1 Incyte Unique DU145 387 010190.1c DU145 388 205311.1 g5931461 e−149 Homo sapiens clone NH0309N08, complete sequence. DU145 389 22429.7 g457372 e−129 dihydroxypolyprenylbenzoate methyltransferase [Rattus DU145 390 59379.1 Incyte Unique DU145 391 236253.1 g6808164 4.00E−83 Human mRNA; cDNA DKFZp761A052 (from clone DKFZp761A052). DU145 392 331033.1 g286012 0 Human mRNA for KIAA0008 gene, complete cds. DU145 393 345533.8c g2385368 0 Human mRNA for Rer1 protein. DU145 394 238342.1 g3483461 0 Human full length insert cDNA clone ZA70C11. DU145 395 28936.1 g3282164 0 Homo sapiens chromosome 5, BAC clone 282B7 (LBNL H192), DU145 396 347865.5 g6807697 2.00E−40 Human mRNA; cDNA DKFZp434A1014 (from clone DKFZp434A1014); partial DU145 397 347865.4 g6807697 1.00E−89 Human mRNA; cDNA DKFZp434A1014 (from clone DKFZp434A1014); partial DU145 398 40057.2 g285946 0 Human mRNA for KIAA0105 gene, complete cds. DU145 399 997301.6 Incyte Unique DU145 400 12235.1 Incyte Unique DU145 401 245496.7 g7298512 4.00E−54 CG10641 gene product [Drosophila melanogaster] DU145 402 983262.3c DU145 403 998084.1 g3483880 0 Human full length insert cDNA clone ZE07G05. DU145 404 238071.2 g5926700 0 Human genomic DNA, chromosome 6p21.3, HLA Class I region, section 12/20. DU145 405 103930.1 Incyte Unique DU145 406 254068.1 g3095056 1.00E−08 Human platelet-activating factor acetylhydrolase gene, promoter region and exon DU145 407 221812.1 g7019862 0 Homo sapiens cDNA FLJ20033 fis, clone COL00106. DU145 408 240129.1 Incyte Unique DU145 409 230297.1c DU145 410 347796.7 g710405 2.8 35 kDa protein [Bartonella henselae] DU145 411 411474.17 g3043687 0 Human mRNA for KIAA0582 protein, partial cds. DU145 412 027434.1c DU145 413 979488.1c DU145 414 213988.1 g7263867 1.00E−134 Human chromosome 14 DNA sequence *** IN PROGRESS *** BAC DU145 TGF&bgr; treated, upregulated 321 405559.1 g897824 1.70E−17 AHNAK gene product DU145 322 16760.1 Incyte Unique DU145 415 263336.62 g187530 0 Human metallothionein-II pseudogene (mt-IIps). DU145 416 153860.6 g4508112 0 Homo sapiens clone RG161A02, complete sequence. DU145 417 154178.1 Incyte Unique DU145 418 337888.3 Incyte Unique DU145 419 240009.2c DU145 420 160011.1 Incyte Unique DU145 421 332919.4 g793840 0 Human mRNA for cytokine inducible nuclear protein. DU145 422 6233.1 Incyte Unique DU145 423 245532.17c g495286 0 Human melanoma differentiation associated (mda-6) mRNA, complete cds. DU145 424 205486.1 Incyte Unique DU145 425 987927.13 g458227 0 Human extracellular protein (S1-5) mRNA, complete cds. DU145 426 25423.3 Incyte Unique DU145 427 1091415.2 g5231136 0 Human angiopoietin-related protein mRNA, complete cds. DU145 428 1091415.16c g5231136 0 Human angiopoietin-related protein mRNA, complete cds. DU145 429 230058.2 g4154282 0 Human xenotropic and polytropic murine leukemia virus receptor (X3) mRNA, DU145 complete cds. 430 444648.9 g187542 0 Human metallothionein (MT)I-F gene, complete cds. DU145 431 464689.22 g4808600 0 Human stearoyl-CoA desaturase (SCD) mRNA, complete cds. DU145 432 464689.15 g4808600 0 Human stearoyl-CoA desaturase (SCD) mRNA, complete cds. DU145 433 334809.3c g36510 0 Human sno oncogene mRNA for snoN protein, ski-related. DU145 434 351241.1 g2935483 4.00E−56 Human minisatellite cebl repeat region. DU145 435 482336.11 g34067 0 Human mRNA fragment for mesothelial type II keratin K7. DU145 436 482336.31 g34067 0 Human mRNA fragment for mesothelial type II keratin K7. DU145 TGF&agr; treated, downregulated 43 60671.7 g3043675 0 Human mRNA for KIAA0576 protein, partial cds. PC3 437 197538.2 g942584 0 Human RAR-responsive (TIG1) mRNA, complete cds. PC3 438 197538.8 g6066619 7.00E−06 latexin [Rattus norvegicus] PC3 439 232048.14 g37638 0 Human mRNA for vascular anticoagulant-beta (VAC-beta). PC3 440 1092381.1 g6634028 0 Human mRNA for KIAA0399 protein, partial cds. PC3 441 903804.1 g5815499 0 Homo sapiens 12p12-27.2-31.7 BAC RPCI11-392P7 (Roswell Park PC3 442 210945.6 g532500 0 Human mRNA for FK506-binding protein 12 kDa (hFKBP-12) homologue, PC3 complete cds. 443 210945.3c g532500 0 Human mRNA for FK506-binding protein 12 kDa (hFKBP-12) homologue, PC3 complete cds. 444 403448.2 g3037018 3.8 NADH dehydrogenase subunit 5 [Bodo saltans] PC3 445 238263.2 Incyte Unique PC3 446 146382.22 g2463541 0 Human mRNA for inositol 1,4,5-trisphosphate 3-kinase isoenzyme, partial cds. PC3 447 146382.25 g2463541 0 Human mRNA for inositol 1,4,5-trisphosphate 3-kinase isoenzyme, partial cds. PC3 448 345705.7 g3047307 0 Human sarcosin mRNA, complete cds. PC3 449 198212.1 g4416529 0 Human skeletal muscle LIM-protein FHL3 mRNA, complete cds. PC3 450 198212.6 g4416529 0 Human skeletal muscle LIM-protein FHL3 mRNA, complete cds. PC3 451 900031.8 g2094872 0 Human DAP-kinase mRNA. PC3 452 900031.4 g2094872 0 Human DAP-kinase mRNA. PC3 453 475365.4 Incyte Unique PC3 454 222810.1 g182662 0 Human N-formylpeptide receptor (fMLP-R98) mRNA, complete cds. PC3 455 1084493.6 g1150990 0 Human receptor tyrosine kinase Flt4 (short form) mRNA, complete cds. PC3 456 979005.2 g4827155 3 bcsCI [Acetobacter xylinus] PC3 457 346686.23 g183046 0 Human granulocyte colony-stimulating factor receptor (G-CSFR-1) mRNA, PC3 complete cds. 458 234568.25 g1469204 0 Human mRNA for KIAA0141 gene, complete cds. PC3 459 234568.17 g1469204 0 Human mRNA for KIAA0141 gene, complete cds. PC3 460 978276.1c PC3 461 26968.1 g2253289 3.00E−09 Human endothelin A receptor gene, 5' flanking region and exon 1. PC3 462 243103.24 g562105 0 Human (dlk) mRNA, complete cds. PC3 463 469883.1 g1663566 0 Human semaphorin (CD100) mRNA, complete cds. PC3 464 903565.14c g2330552 0 Human mRNA for PACE4A-II, complete cds. PC3 465 227932.2 g2264346 1.00E−141 GOK [Homo sapiens] PC3 466 903691.6 g439295 0 Human garp gene mRNA, complete cds. PC3 467 012995.19c g6682360 0 Human talin mRNA, complete cds. PC3 468 407938.5 g2765321 0 Human mRNA for RB18A protein. PC3 469 233778.1 g487808 0 Human cell surface protein (NKG7) mRNA, complete cds. PC3 470 474630.19 g33910 0 Human mRNA for integrin beta(4)subunit. PC3 471 217319.7 g1033874 7.00E−11 Human CpG island DNA genomic Msel fragment, clone 53c10, reverse read PC3 cpg53c10.rtlb. 472 1092445.1 g5915897 0 Human zinc finger protein 42 (ZNF42) mRNA, alternate transcript, complete cds. PC3 473 199433.3 g4323621 0 Human intracellular chloride channel CLIC3 (CLIC3) mRNA, complete cds. PC3 474 228860.6 g2865608 0 Human homogentisate 1,2-dioxygenase (AKU) mRNA, complete cds. PC3 475 228860.4c g2130646 0 Human homogentisate 1,2-dioxygenase gene, complete cds. PC3 476 410688.3 g1405359 9.00E−89 Human fetal brain (239FB) mRNA, from the WAGR region, complete cds. PC3 477 347005.5 g35787 0 Human HPTP beta mRNA for protein tyrosine phosphatase beta. PC3 478 334621.13 g3599961 5.00E−90 Human h-bcs1 (BCS1) mRNA, nuclear gene encoding mitochondrial protein, PC3 complete cds. 479 28180.1 Incyte Unique PC3 480 252570.6c g3820529 7.2 protein kinase homolog; AbiBL11 [Bacillus licheniformis] PC3 481 466402.116 g435059 0 Human Golli-mbp gene, complete cds. PC3 482 1096160.26c g561542 0 Human protein kinase (zpk) mRNA, complete cds. PC3 483 899612.2 g4884371 0 Human mRNA; cDNA DKFZp586A0522 (from clone DKFZp586A0522); partial PC3 484 254107.1 g220126 0 Human gene for thrombomodulin precursor, complete cds. PC3 485 208328.1 g2546963 0 Human mRNA for diubiquitin. PC3 486 130157.2 g5870324 1.00E−61 supported by human EST AA331940 (NID: g1984182), mouse EST AA387866 PC3 (NID: 2040812), and Genscan [Homo sapiens] 487 985824.2c PC3 488 233089.1 g7242976 0 Homo sapiens mRNA for KIAA1311 protein, partial cds. PC3 489 336256.1 g963053 0 Human mRNA for membrane-type matrix metalloprotease 1. PC3 490 979616.2 g563140 2.4 cytochrome c oxidase subunit 1 [Trypanoplasma borreli] PC3 491 351432.3 g1809029 0 Human AE2 anion exchanger (SLC4A2) mRNA, complete cds. PC3 492 83495.1 Incyte Unique PC3 493 332595.1 g2979421 0 Human mRNA for PCDH7 (BH-Pcdh)c, complete cds. PC3 494 481251.3 g5917667 0 Human cysteine-rich hydrophobic 2 CHIC2 mRNA, complete cds. PC3 495 25612.1 Incyte Unique PC3 496 96700.1 Incyte Unique PC3 497 230048.1 Incyte Unique PC3 498 073621.3c g6330384 0 Human mRNA for KIAA1197 protein, partial cds. PC3 499 344582.19 g4106877 0 Human decoy receptor 3 (DcR3) mRNA, complete cds. PC3 500 997526.4 g5102629 0 Novel Human gene mapping to chomosome 22. PC3 501 997526.1 g3483012 0 Human mRNA for HMGBCG protein. PC3
[0178] 2 TABLE 2 SEQ TEMPLATE ID NO ID START STOP FRAME Pfam Description E-Value 3 977955.7 984 1133 forward 3 Phorbol esters/diacylglycerol binding domain (C1 domain) 2.40E−21 3 977955.7 1440 1796 forward 3 PH domain 7.10E−12 3 977955.7 1920 2690 forward 3 Eukaryotic protein kinase domain 5.70E−76 4 350754.2 857 1123 forward 2 Transglutaminase-like superfamily 1.40E−46 4 350754.2 1457 2119 forward 2 Transglutaminase family 3.20E−50 4 350754.2 56 415 forward 2 Transglutaminase family 2.20E−63 5 235191.4 180 617 forward 3 Ubiquitin-conjugating enzyme 3.30E−57 7 1099593.2 325 447 forward 1 Armadillo/beta-catenin-like repeats 2.20E−07 11 347915.14 1585 1911 forward 1 PH domain 2.00E−14 11 347915.14 2191 2436 forward 1 Src homology domain 2 1.90E−21 12 997395.1 2027 2143 forward 2 WD domain, G-beta repeat 2.10E−05 16 404155.2 2133 2417 forward 3 Cyclic nucleotide-binding domain 6.20E−30 16 404155.2 2587 3207 forward 1 Eukaryotic protein kinase domain 3.30E−71 16 404155.2 2505 2669 forward 3 Eukaryotic protein kinase domain 2.20E−06 16 404155.2 3208 3306 forward 1 Protein kinase C terminal domain 1.30E−04 19 345860.21 459 1109 forward 3 Papain family cysteine protease 2.80E−133 20 199101.1 612 1172 forward 3 Glycosyl transferases 9.60E−18 20 199101.1 1554 1940 forward 3 Similarity to lectin domain of ricin beta-chain, 3 copies. 3.00E−09 22 475146.3 197 1567 forward 2 Hydroxymethylglutaryl-coenzyme A synthase 0.00E+00 24 255115.2 91 1605 forward 1 UDP-glucoronosyl and UDP-glucosyl transferases 0.00E+00 25 255115.4 121 1023 forward 1 UDP-glucoronosyl and UDP-glucosyl transferases 1.70E−177 25 255115.4 977 1573 forward 2 UDP-glucoronosyl and UDP-glucosyl transferases 2.60E−158 29 978047.1 613 1287 forward 1 alpha/beta hydrolase fold 1.20E−04 37 399067.1 1426 1611 forward 1 Immunoglobulin domain 1.50E−08 37 399067.1 538 609 forward 1 Leucine Rich Repeat 1.40E−04 37 399067.1 1219 1377 forward 1 Leucine rich repeat C-terminal domain 9.90E−13 37 399067.1 199 321 forward 1 Leucine rich repeat N-terminal domain 6.70E−04 41 255824.52 642 1244 forward 3 Fructose-bisphosphate aldolase class-I 5.60E−149 41 255824.52 1237 1644 forward 1 Fructose-bisphosphate aldolase class-I 7.30E−96 44 376085.9 1596 1664 forward 3 Zinc finger, C2H2 type 2.10E−05 45 404467.1 6 764 forward 3 7 transmembrane receptor (Secretin family) 6.30E−09 49 391406.5 2183 2299 forward 2 WD domain, G-beta repeat 1.60E−13 49 391406.5 1128 1244 forward 3 WD domain, G-beta repeat 1.60E−13 50 391406.24 349 465 forward 1 WD domain, G-beta repeat 1.60E−13 50 391406.24 87 206 forward 3 WD domain, G-beta repeat 2.10E−07 51 474588.21 2263 2460 forward 1 RNA recognition motif. (a.k.a. RRM, RBD, or RNP domain) 9.20E−21 51 474588.21 1863 2075 forward 3 RNA recognition motif. (a.k.a. RRM, RBD, or RNP domain) 6.90E−09 53 246862.9 1391 1489 forward 2 ATP synthase Alpha chain, C terminal 1.20E−22 53 246862.9 1315 1401 forward 1 ATP synthase Alpha chain, C terminal 4.10E−20 53 246862.9 347 1315 forward 2 ATP synthase alpha/beta family 1.10E−87 53 246862.9 307 1071 forward 1 ATP synthase alpha/beta family 9.60E−10 54 246862.17 1428 1832 forward 3 ATP synthase Alpha chain, C terminal 4.30E−106 54 246862.17 303 1427 forward 3 ATP synthase alpha/beta family 1.60E−187 56 236480.3 2154 2264 forward 3 WD domain, G-beta repeat 7.30E−08 57 314831.5 841 2037 forward 1 Elongation factor Tu family 6.10E−142 58 391940.1 1300 2004 forward 1 alpha/beta hydrolase fold 1.70E−31 59 454958.13 266 799 forward 2 Tissue inhibitor of metalloproteinases 1.80E−141 61 238203.11 89 3544 forward 2 Vinculin family 9.30E−281 61 238203.11 828 3182 forward 3 Vinculin family 4.50E−110 62 994057.21 277 1155 forward 1 Ser/Thr protein phosphatase 6.10E−202 63 346730.5 71 1300 forward 2 Monocarboxylate transporter 1.10E−48 66 482336.2 768 1706 forward 3 Intermediate filament proteins 5.50E−167 67 482336.14 654 1592 forward 3 Intermediate filament proteins 1.30E−167 69 300294.3 589 1392 forward 1 SRP54-type protein 4.00E−141 71 475028.7 203 721 forward 2 ADP-ribosylation factor family 8.10E−06 71 475028.7 233 853 forward 2 Ras family 2.90E−92 72 481223.3 430 636 forward 1 HMG (high mobility group) box 3.80E−31 74 234340.7 1164 1412 forward 3 Ets-domain 1.80E−53 77 317586.1 471 1253 forward 3 N-acetyltransferase 1.40E−208 86 1063057.1 153 221 forward 3 Zinc finger, C2H2 type 1.60E−06 87 199905.1 48 200 forward 3 Laminin EGF-like (Domains III and V) 8.90E−13 90 330878.6 178 2286 forward 1 Cullin family 2.40E−222 91 331793.11 175 819 forward 1 emp24/gp25L/p24 family 4.50E−55 97 977667.1 273 1013 forward 3 7 transmembrane receptor (rhodopsin family) 4.40E−77 99 1136709.5 284 601 forward 2 Vertebrate galactoside-binding lectins 1.60E−58 100 1136709.6 137 454 forward 2 Vertebrate galactoside-binding lectins 1.80E−57 101 382293.16 756 1094 forward 3 CUB domain 4.00E−42 101 382293.16 636 746 forward 3 EGF-like domain 1.40E−06 101 382293.16 1308 1496 forward 3 Sushi domain (SCR repeat) 1.20E−12 101 382293.16 1545 2258 forward 3 Trypsin 3.30E−68 102 382293.15 988 1326 forward 1 CUB domain 4.00E−42 102 382293.15 868 978 forward 1 EGF-like domain 1.40E−06 102 382293.15 1567 1755 forward 1 Sushi domain (SCR repeat) 1.20E−12 102 382293.15 1804 2517 forward 1 Trypsin 3.30E−68 104 235943.36 184 531 forward 1 SEA domain 2.40E−29 105 401434.1 410 604 forward 2 DnaJ domain 9.70E−35 106 234107.2 1162 1311 forward 1 Immunoglobulin domain 4.60E−08 106 234107.2 177 422 forward 3 Immunoglobulin domain 2.50E−05 109 441298.14 26 175 forward 2 PHD-finger 1.40E−11 113 230936.6 184 996 forward 1 Ribosomal RNA adenine dimethylases 8.10E−32 116 405158.1 23 121 forward 2 Ank repeat 1.20E−04 116 405158.1 756 986 forward 3 IBR domain 9.00E−15 118 480187.81 305 538 forward 2 Immunoglobulin domain 2.40E−08 119 62042.4 247 492 forward 1 Fibronectin type III domain 1.40E−11 120 1087696.7 179 769 forward 2 short chain dehydrogenase 9.20E−45 121 259805.28 328 1083 forward 1 Eukaryotic protein kinase domain 5.20E−100 121 259805.28 1141 1257 forward 1 UBA domain 6.00E−06 123 986565.15 241 1533 forward 1 Reduced folate carrier 9.10E−143 124 978378.3 361 999 forward 1 TBC domain 1.90E−37 127 238635.1 123 296 forward 3 Immunoglobulin domain 2.60E−09 128 1080496.1 1256 1324 forward 2 Zinc finger, C2H2 type 9.80E−04 129 351204.2 190 777 forward 1 Trypsin 5.70E−76 133 474868.2 209 403 forward 2 DnaJ domain 1.20E−36 133 474868.2 866 1234 forward 2 DnaJ C terminal region 2.50E−18 140 411296.2 1186 1923 forward 1 Eukaryotic protein kinase domain 9.90E−73 140 411296.2 832 1080 forward 1 Src homology domain 2 2.00E−50 140 411296.2 643 810 forward 1 SH3 domain 2.70E−25 150 346511.4 1129 1449 forward 1 Nucleotidyltransferase domain 8.90E−17 151 346511.6 1124 1444 forward 2 Nucleotidyltransferase domain 8.90E−17 153 234056.5 162 899 forward 3 Transmembrane 4 family 1.60E−47 156 988653.1 1295 1369 forward 2 Zinc finger, C2H2 type 1.00E−06 157 470468.26 726 1472 forward 3 Phosphorylase family 1.20E−04 161 1094412.1 298 2118 forward 1 Hsp70 protein 0.00E+00 162 1094107.1 293 2104 forward 2 Hsp70 protein 4.80E−204 162 1094107.1 813 2066 forward 3 Hsp70 protein 1.30E−31 164 12178.2 15 248 forward 3 E1-E2 ATPase 1.20E−09 165 100653.3 313 486 forward 1 Helix-loop-helix DNA-binding domain 3.20E−13 171 331065.2 244 510 forward 1 Caspase recruitment domain 3.70E−32 171 331065.2 1226 1507 forward 2 ICE-like protease (caspase) p10 domain 1.60E−50 171 331065.2 739 1128 forward 1 ICE-like protease (caspase) p20 domain 9.40E−77 172 331065.5 180 446 forward 3 Caspase recruitment domain 3.70E−32 172 331065.5 1143 1424 forward 3 ICE-like protease (caspase) p10 domain 1.60E−50 172 331065.5 675 1064 forward 3 ICE-like protease (caspase) p20 domain 1.50E−81 173 252782.4 644 793 forward 2 F-box domain. 2.20E−04 176 200163.2 468 1289 forward 3 Cell division protein 1.90E−119 178 1328237.4 780 1508 forward 3 Eukaryotic protein kinase domain 4.60E−54 179 333965.1 1179 1979 forward 3 Eukaryotic protein kinase domain 2.50E−59 179 333965.1 1980 2066 forward 3 Protein kinase C terminal domain 2.30E−08 180 1328236.5 112 672 forward 1 Ras family 5.90E−87 181 995534.3 686 811 forward 2 WD domain, G-beta repeat 5.40E−05 182 1098887.1 325 612 forward 1 SCAN domain 7.80E−70 182 1098887.1 1192 1260 forward 1 Zinc finger, C2H2 type 4.60E−07 184 470023.2 456 815 forward 3 Serpins (serine protease inhibitors) 2.40E−68 185 989966.8 424 1611 forward 1 N2,N2-dimethylguanosine tRNA methyltransferase 2.60E−221 185 989966.8 1872 1958 forward 3 Zinc finger C-x8-C-x5-C-x3-H type (and similar). 2.90E−04 186 233925.5 571 783 forward 1 RNA recognition motif. (a.k.a. RRM, RBD, or RNP domain) 6.50E−20 187 405145.5 1112 1579 forward 2 GAF domain 1.50E−28 187 405145.5 2456 3172 forward 2 3'5'-cyclic nucleotide phosphodiesterase 6.60E−96 188 261982.8 6390 6641 forward 3 Helicases conserved C-terminal domain 2.00E−15 188 261982.8 4863 5843 forward 3 SNF2 and others N-terminal domain 7.70E−139 193 898850.21 3205 3297 forward 1 Tau and MAP proteins, tubulin-binding 1.60E−16 196 318000.4 225 755 forward 3 ADP-ribosylation factor family 1.20E−39 196 318000.4 267 875 forward 3 Ras family 7.20E−08 197 977887.1 2043 2825 forward 3 Uncharacterized protein family 8.30E−97 198 1094199.1 513 707 forward 3 PAS domain 1.50E−08 202 113621.5 89 625 forward 2 Tissue inhibitor of metalloproteinases 1.20E−144 203 401532.2 1067 1882 forward 2 ABC transporter transmembrane region. 4.40E−44 203 401532.2 4082 4633 forward 2 ABC transporter 3.40E−46 207 475819.14 656 769 forward 2 Zinc finger, C3HC4 type (RING finger) 1.80E−06 207 475819.14 656 769 forward 2 Zinc finger, C3HC4 type (RING finger) 1.80E−06 208 241384.3 78 1982 forward 3 Glycosyl hydrolases family 35 0.00E+00 210 244200.1 394 948 forward 1 Glycosyl transferases 2.40E−52 210 244200.1 1315 1692 forward 1 Similarity to lectin domain of ricin beta-chain, 3 copies. 3.20E−45 214 903091.31 994 1131 forward 1 Zinc finger present in dystrophin, CBP/p300 3.30E−07 216 903091.16 876 1013 forward 3 Zinc finger present in dystrophin, CBP/p300 3.30E−07 218 475473.1 6003 6053 forward 3 Neuraxin and MAP1B proteins 1.20E−08 223 903338.12 142 300 forward 1 Defensin propeptide 3.00E−25 223 903338.12 337 423 forward 1 Mammalian defensin 3.10E−14 225 1040667.52 1868 2074 forward 2 RNA recognition motif. (a.k.a. RRM, RBD, or RNP domain) 2.40E−04 227 218524.4 596 1345 forward 2 7 transmembrane receptor (rhodopsin family) 7.10E−94 228 346673.1 533 727 forward 2 Double-stranded RNA binding motif 2.60E−06 230 197086.1 1317 1460 forward 3 GRIP domain 2.10E−07 231 348143.7 677 796 forward 2 UBA domain 9.20E−13 231 348143.7 206 649 forward 2 Ubiquitin-conjugating enzyme 5.50E−79 233 233003.6 509 604 forward 2 Ubiquitin carboxyl-terminal hydrolases family 2 8.60E−06 233 233003.6 1238 1504 forward 2 Ubiquitin carboxyl-terminal hydrolase family 2 3.30E−19 235 217860.1 559 900 forward 1 BTB/POZ domain 4.40E−16 236 216189.2 431 898 forward 2 BTG1 family 3.00E−103 238 382906.16 182 226 forward 2 Transmembrane 4 family 4.40E−04 244 413835.5 250 633 forward 1 BTB/POZ domain 3.60E−23 244 413835.5 1318 1386 forward 1 Zinc finger, C2H2 type 7.80E−06 246 474673.1 4099 4554 forward 1 RhoGAP domain 2.60E−54 247 480286.1 322 654 forward 1 Core histone H2A/H2B/H3/H4 2.50E−16 247 480286.1 347 679 forward 2 Core histone H2A/H2B/H3/H4 1.20E−14 252 253946.17 985 1254 forward 1 Fibronectin type III domain 3.00E−14 261 82168.5 9273 9422 forward 3 Leucine rich repeat C-terminal domain 6.40E−12 261 82168.5 9564 9980 forward 3 TIR domain 6.60E−42 263 412661.2 269 463 forward 2 FHA domain 4.30E−21 267 385608.2 251 991 forward 2 Transmembrane 4 family 2.30E−160 279 82154.24 1684 1800 forward 1 WD domain, G-beta repeat 7.70E−04 279 82154.24 1684 1800 forward 1 WD domain, G-beta repeat 7.70E−04 283 90710.1 155 223 forward 2 Zinc finger, C2H2 type 1.20E−04 290 350440.15 960 2684 forward 3 E1-E2 ATPase 9.00E−59 290 350440.15 2599 2952 forward 1 E1-E2 ATPase 8.60E−42 304 400280.3 600 803 forward 3 RNA recognition motif. (a.k.a. RRM, RBD, or RNP domain) 3.80E−09 318 481847.1 202 318 forward 1 EGF-like domain 5.20E−10 333 433569.1 450 1688 forward 3 Sulfatase 4.70E−103 333 433569.1 260 1687 forward 2 Sulfatase 5.50E−39 341 405041.1 435 533 forward 3 Ank repeat 2.00E−08 342 474266.2 424 522 forward 1 Ank repeat 2.30E−07 345 195199.1 886 984 forward 1 Ank repeat 1.10E−04 348 256871.2 609 707 forward 3 Ank repeat 2.00E−05 348 256871.2 609 707 forward 3 Ank repeat 2.00E−05 356 344775.3 604 993 forward 1 SPRY domain 1.80E−19 358 416874.3 313 558 forward 1 Kringle domain 7.10E−37 358 416874.3 640 1362 forward 1 Trypsin 1.70E−102 362 336953.7 2154 2882 forward 3 Biotin protein ligase 2.30E−11 363 444771.2 2 352 forward 2 Elongation factor G C-terminus 2.00E−04 364 449173.16 641 730 forward2 KH domain 3.10E−04 367 252747.27 2298 2567 forward 3 C2 domain 8.50E−17 369 1000084.27 152 1423 forward 2 Tubulin/FtsZ family 2.40E−279 370 205607.5 1849 2733 forward 1 Phosphatidylinositol 3- and 4-kinases 7.40E−116 370 205607.5 139 468 forward 1 C2 domain 3.10E−44 370 205607.5 925 1668 forward 1 Phosphoinositide 3-kinase family, accessory domain (PIK dom 2.10E−58 374 474724.5 340 780 forward 1 Eukaryotic protein kinase domain 6.70E−25 394 238342.1 140 502 forward 2 BAH domain 2.50E−16 411 411474.17 174 719 forward 3 ADP-ribosylation factor family 7.60E−08 411 411474.17 231 815 forward 3 Ras family 1.10E−112 415 263336.62 272 454 forward 2 Metallothionein 2.10E−25 421 332919.4 703 801 forward 1 Ank repeat 9.40E−08 425 987927.13 681 788 forward 3 EGF-like domain 2.10E−04 427 1091415.2 775 1425 forward 1 Fibrinogen beta and gamma chains, C-terminal globular domai 5.60E−63 430 444648.9 366 548 forward 3 Metallothionein 1.20E−24 431 464689.22 608 1342 forward 2 Fatty acid desaturase 1.20E−163 435 482336.11 95 310 forward 2 Intermediate filament proteins 1.50E−21 442 210945.6 155 481 forward 2 FKBP-type peptidyl-prolyl cis-trans isomerases 1.90E−52 448 345705.7 232 573 forward 1 BTB/POZ domain 1.30E−28 448 345705.7 1525 1668 forward 1 Kelch motif 2.50E−09 449 198212.1 816 986 forward 3 LIM domain containing proteins 3.00E−15 450 198212.6 635 805 forward 2 LIM domain containing proteins 3.00E−15 450 198212.6 277 453 forward 1 LIM domain containing proteins 8.60E−07 450 198212.6 801 968 forward 3 LIM domain containing proteins 2.10E−06 452 900031.4 2003 2101 forward 2 Ank repeat 1.60E−05 452 900031.4 4406 4663 forward 2 Death domain 9.40E−20 452 900031.4 509 1297 forward 2 Eukaryotic protein kinase domain 4.60E−87 453 475365.4 607 1455 forward 1 Zinc carboxypeptidase 1.30E−111 454 222810.1 384 965 forward 3 7 transmembrane receptor (rhodopsin family) 2.60E−34 454 222810.1 206 409 forward 2 7 transmembrane receptor (rhodopsin family) 1.00E−15 455 1084493.6 516 995 forward 3 Eukaryotic protein kinase domain 1.00E−47 455 1084493.6 31 258 forward 1 Eukaryotic protein kinase domain 2.70E−08 457 346686.23 1881 2129 forward 3 Fibronectin type III domain 1.00E−06 462 243103.24 683 778 forward 2 EGF-like domain 7.40E−09 463 469883.1 2253 2426 forward 3 Immunoglobulin domain 5.20E−08 463 469883.1 2052 2210 forward 3 Plexin repeat 4.80E−09 463 469883.1 696 1994 forward 3 Sema domain 1.40E−207 466 903691.6 261 344 forward 3 Leucine rich repeat N-terminal domain 5.60E−04 470 474630.19 4403 4657 forward 2 Fibronectin type III domain 1.80E−25 470 474630.19 3493 3738 forward 1 Fibronectin type III domain 3.70E−19 470 474630.19 264 1520 forward 3 Integrins, beta chain 6.3e−317 472 1092445.1 399 686 forward 3 SCAN domain 5.50E−58 472 1092445.1 1437 1505 forward 3 Zinc finger, C2H2 type 2.40E−07 477 347005.5 361 603 forward 1 Fibronectin type III domain 9.60E−18 477 347005.5 5206 5913 forward 1 Protein-tyrosine phosphatase 1.50E−140 481 466402.116 657 1082 forward 3 Myelin basic protein 9.50E−08 484 254107.1 1446 1553 forward 3 EGF-like domain 3.60E−05 485 208328.1 58 273 forward 1 Ubiquitin family 7.90E−09 489 336256.1 1478 1618 forward 2 Hemopexin 2.60E−16 489 336256.1 347 901 forward 2 Matrixin 1.10E−56 491 351432.3 2537 4858 forward 2 HCO3- transporter family 0.00E+00 493 332595.1 320 616 forward 2 Cadherin domain 1.20E−09 493 332595.1 1 261 forward 1 Cadherin domain 7.60E−07 499 344582.19 375 497 forward 3 TNFR/NGFR cysteine-rich region 6.80E−05 500 997526.4 5 202 forward 2 HMG (high mobility group) box 8.20E−13
[0179] 3 TABLE 3 SEQ ID- NO TEMPLATE ID START STOP FRAME DOMAIN 1 441269.2 928 1008 forward 1 SP 2 3161.5 571 651 forward 1 TM 2 3161.5 1426 1506 forward 1 TM 2 3161.5 388 471 forward 1 SP 2 3161.5 382 468 forward 1 TM 3 977955.7 174 263 forward 3 SP 4 350754.2 651 749 forward 3 SP 4 350754.2 270 362 forward 3 SP 6 1099593.13 730 810 forward 1 SP 6 1099593.13 2130 2210 forward 3 TM 7 1099593.2 161 259 forward 2 SP 11 347915.14 4742 4840 forward 2 SP 12 997395.1 3240 3323 forward 3 SP 12 997395.1 2100 2180 forward 3 SP 12 997395.1 3187 3291 forward 1 SP 13 332783.1 1164 1244 forward 3 TM 13 332783.1 1434 1511 forward 3 TM 14 422289.1 2247 2327 forward 3 TM 14 422289.1 2059 2142 forward 1 SP 15 899410.5 1077 1163 forward 3 TM 15 899410.5 2158 2244 forward 1 SP 15 899410.5 350 430 forward 2 SP 16 404155.2 265 348 forward 1 TM 19 345860.21 772 864 forward 1 SP 23 474069.8 970 1050 forward 1 TM 26 216331.1 1465 1551 forward 1 TM 27 482517.3 1939 2019 forward 1 TM 27 482517.3 148 228 forward 1 TM 27 482517.3 350 424 forward 2 TM 27 482517.3 1170 1253 forward 3 SP 27 482517.3 1490 1564 forward 2 TM 27 482517.3 1753 1833 forward 1 TM 27 482517.3 300 377 forward 3 TM 29 978047.1 1784 1861 forward 2 TM 29 978047.1 1834 1914 forward 1 TM 29 978047.1 1094 1180 forward 2 SP 33 898068.6 446 532 forward 2 SP 35 903104.8 1628 1717 forward 2 TM 36 412065.21 1549 1635 forward 1 SP 36 412065.21 1478 1579 forward 2 SP 37 399067.1 2363 2446 forward 2 TM 37 399067.1 109 189 forward 1 SP 38 237709.11 301 384 forward 1 SP 39 237709.5 766 849 forward 1 SP 40 216437.4 432 527 forward 3 SP 40 216437.4 479 559 forward 2 TM 42 27798.1 57 137 forward 3 TM 42 27798.1 2134 2226 forward 1 SP 42 27798.1 1483 1566 forward 1 SP 44 376085.9 656 751 forward 2 SP 46 903508.12 2552 2632 forward 2 TM 49 391406.5 463 546 forward 1 SP 49 391406.5 2070 2153 forward 3 SP 50 391406.24 208 291 forward 1 SP 51 474588.21 1315 1392 forward 1 TM 53 246862.9 834 932 forward 3 SP 54 246862.17 1393 1473 forward 1 SP 54 246862.17 934 1032 forward 1 SP 54 246862.17 1573 1662 forward 1 SP 57 314831.5 1070 1159 forward 2 SP 58 391940.1 1112 1192 forward 2 SP 58 391940.1 5869 5946 forward 1 TM 58 391940.1 4958 5038 forward 2 SP 58 391940.1 6060 6146 forward 3 SP 59 454958.13 214 291 forward 1 SP 61 238203.11 2656 2733 forward 1 SP 61 238203.11 4781 4864 forward 2 TM 61 238203.11 4074 4154 forward 3 TM 62 994057.21 819 902 forward 3 SP 62 994057.21 3427 3498 forward 1 TM 62 994057.21 1776 1862 forward 3 TM 66 482336.2 1051 1137 forward 1 SP 66 482336.2 1702 1785 forward 1 SP 67 482336.14 934 1020 forward 1 SP 67 482336.14 1585 1668 forward 1 SP 68 66522.1 294 380 forward 3 TM 68 66522.1 31 108 forward 1 SP 69 300294.3 1976 2053 forward 2 SP 71 475028.7 1972 2061 forward 1 SP 72 481223.3 2466 2561 forward 3 SP 72 481223.3 1326 1406 forward 3 SP 76 255778.11 1290 1376 forward 3 TM 80 201342.4 528 608 forward 3 TM 81 204542.1 9 92 forward 3 SP 83 204386.1 1466 1549 forward 2 SP 83 204386.1 1553 1642 forward 2 SP 84 335086.1 2638 2718 forward 1 TM 84 335086.1 376 462 forward 1 TM 86 1063057.1 293 382 forward 2 SP 87 199905.1 4765 4851 forward 1 TM 87 199905.1 2052 2129 forward 3 SP 87 199905.1 817 900 forward 1 SP 87 199905.1 981 1052 forward 3 TM 87 199905.1 236 340 forward 2 SP 88 11329.1 185 265 forward 2 TM 90 330878.6 2974 3066 forward 1 TM 90 330878.6 2328 2405 forward 3 TM 91 331793.11 3030 3107 forward 3 TM 91 331793.11 139 219 forward 1 SP 95 1095702.14 256 342 forward 1 SP 97 977667.1 234 311 forward 3 TM 101 382293.16 2167 2316 forward 1 SP 101 382293.16 5189 5278 forward 2 SP 101 382293.16 6113 6205 forward 2 SP 101 382293.16 598 687 forward 1 SP 101 382293.16 6017 6097 forward 2 SP 101 382293.16 596 682 forward 2 SP 102 382293.15 2423 2572 forward 2 SP 102 382293.15 827 916 forward 2 SP 102 382293.15 816 902 forward 3 SP 104 235943.36 572 664 forward 2 SP 104 235943.36 52 132 forward 1 SP 106 234107.2 54 134 forward 3 SP 108 482423.1 842 931 forward 2 SP 108 482423.1 389 466 forward 2 SP 109 441298.14 4843 4923 forward 1 SP 109 441298.14 480 569 forward 3 SP 109 441298.14 4439 4519 forward 2 SP 112 992317.12 219 323 forward 3 SP 113 230936.6 2690 2776 forward 2 TM 113 230936.6 2433 2528 forward 3 SP 115 984900.1 464 541 forward 2 TM 116 405158.1 3164 3238 forward 2 TM 116 405158.1 4889 4972 forward 2 SP 116 405158.1 4318 4404 forward 1 TM 116 405158.1 5463 5540 forward 3 TM 120 1087696.7 56 142 forward 2 SP 122 399128.1 27 116 forward 3 TM 123 986565.15 1255 1335 forward 1 TM 123 986565.15 2254 2331 forward 1 TM 123 986565.15 1121 1204 forward 2 SP 123 986565.15 2148 2225 forward 3 TM 123 986565.15 2493 2570 forward 3 TM 123 986565.15 1430 1522 forward 2 SP 126 233594.4 613 699 forward 1 SP 128 1080496.1 1278 1373 forward 3 SP 129 351204.2 554 631 forward 2 SP 129 351204.2 35 124 forward 2 SP 132 427529.9 145 225 forward 1 SP 133 474868.2 459 542 forward 3 SP 133 474868.2 382 468 forward 1 SP 134 1095839.17 406 501 forward 1 SP 135 1095839.1 29 142 forward 2 SP 135 1095839.1 1105 1182 forward 1 TM 135 1095839.1 1813 1890 forward 1 TM 136 244251.4 239 325 forward 2 SP 136 244251.4 720 800 forward 3 SP 136 244251.4 70 150 forward 1 TM 136 244251.4 651 734 forward 3 TM 138 234157.3 1914 2006 forward 3 SP 142 407260.2 296 382 forward 2 SP 145 231270.1 1138 1227 forward 1 SP 148 404197.4 5001 5081 forward 3 SP 148 404197.4 461 562 forward 2 SP 148 404197.4 1523 1615 forward 2 SP 150 346511.4 3324 3428 forward 3 SP 152 331233.2 19 114 forward 1 SP 154 238118.1 21 98 forward 3 TM 155 233596.5 1762 1839 forward 1 TM 155 233596.5 2332 2421 forward 1 SP 156 988653.1 1080 1166 forward 3 SP 156 988653.1 3422 3502 forward 2 TM 157 470468.26 913 999 forward 1 SP 159 903479.3 2576 2659 forward 2 TM 159 903479.3 2571 2663 forward 3 TM 159 903479.3 6877 6951 forward 1 TM 159 903479.3 2017 2094 forward 1 TM 159 903479.3 2563 2640 forward 1 TM 159 903479.3 5437 5514 forward 1 TM 159 903479.3 5549 5632 forward 2 TM 161 1094412.1 179 259 forward 2 SP 161 1094412.1 1565 1651 forward 2 SP 162 1094107.1 179 259 forward 2 SP 162 1094107.1 1510 1596 forward 1 SP 162 1094107.1 669 794 forward 3 SP 164 12178.2 387 461 forward 3 TM 164 12178.2 430 510 forward 1 SP 166 978478.4 791 862 forward 2 TM 167 85942.2 285 371 forward 3 SP 170 283762.2 4645 4725 forward 1 TM 170 283762.2 4511 4600 forward 2 SP 171 331065.2 72 161 forward 3 SP 173 252782.4 3071 3154 forward 2 SP 173 252782.4 1839 1922 forward 3 TM 173 252782.4 1371 1451 forward 3 TM 173 252782.4 1800 1883 forward 3 SP 174 229068.1 648 731 forward 3 SP 175 407450.7 1416 1502 forward 3 TM 176 200163.2 367 450 forward 1 SP 176 200163.2 3400 3474 forward 1 TM 176 200163.2 3730 3828 forward 1 SP 178 1328237.4 685 777 forward 1 SP 178 1328237.4 2079 2159 forward 3 TM 178 1328237.4 2006 2089 forward 2 TM 178 1328237.4 2374 2451 forward 1 TM 179 333965.1 1357 1440 forward 1 SP 179 333965.1 435 542 forward 3 SP 180 1328236.5 1014 1091 forward 3 TM 181 995534.3 708 797 forward 3 SP 184 470023.2 306 389 forward 3 SP 186 233925.5 1436 1510 forward 2 TM 186 233925.5 440 523 forward 2 SP 186 233925.5 1901 1984 forward 2 TM 186 233925.5 3519 3596 forward 3 TM 187 405145.5 6884 6967 forward 2 TM 187 405145.5 6924 7043 forward 3 SP 187 405145.5 1491 1574 forward 3 SP 188 261982.8 7984 8061 forward 1 TM 188 261982.8 9033 9110 forward 3 TM 188 261982.8 9430 9522 forward 1 TM 188 261982.8 7748 7828 forward 2 TM 189 246285.1 833 937 forward 2 SP 190 228511.1 4994 5074 forward 2 TM 190 228511.1 4777 4854 forward 1 SP 190 228511.1 1557 1640 forward 3 TM 191 40290.1 656 736 forward 2 TM 193 898850.21 4370 4456 forward 2 TM 193 898850.21 3429 3518 forward 3 SP 194 201356.1 923 1006 forward 2 TM 196 318000.4 2025 2102 forward 3 SP 196 318000.4 1684 1764 forward 1 TM 197 977887.1 1426 1536 forward 1 SP 197 977887.1 3731 3820 forward 2 SP 198 1094199.1 1516 1641 forward 1 SP 198 1094199.1 4082 4174 forward 2 SP 202 113621.5 1299 1382 forward 3 SP 202 113621.5 17 94 forward 2 SP 203 401532.2 3138 3251 forward 3 SP 203 401532.2 5214 5291 forward 3 TM 203 401532.2 1914 1991 forward 3 SP 203 401532.2 1415 1498 forward 2 SP 203 401532.2 3449 3532 forward 2 TM 203 401532.2 4014 4103 forward 3 SP 205 351209.14 256 336 forward 1 SP 206 351209.16 2134 2223 forward 1 SP 206 351209.16 285 368 forward 3 SP 206 351209.16 266 352 forward 2 SP 208 241384.3 1003 1098 forward 1 SP 208 241384.3 45 146 forward 3 SP 210 244200.1 2306 2389 forward 2 TM 210 244200.1 2136 2219 forward 3 TM 216 903091.16 1420 1506 forward 1 SP 216 903091.16 1768 1854 forward 1 SP 218 475473.1 5134 5223 forward 1 SP 218 475473.1 7072 7152 forward 1 SP 219 354430.4 4937 5020 forward 2 SP 219 354430.4 6314 6409 forward 2 SP 219 354430.4 469 546 forward 1 SP 219 354430.4 5436 5540 forward 3 SP 223 903338.12 115 198 forward 1 SP 225 1040667.52 3266 3349 forward 2 TM 225 1040667.52 666 749 forward 3 SP 225 1040667.52 3309 3395 forward 3 SP 225 1040667.52 510 590 forward 3 TM 225 1040667.52 3408 3491 forward 3 SP 227 218524.4 1130 1210 forward 2 SP 227 218524.4 1350 1433 forward 3 SP 227 218524.4 554 634 forward 2 TM 229 58775.1 2 88 forward 2 SP 231 348143.7 2697 2771 forward 3 TM 231 348143.7 4440 4526 forward 3 TM 231 348143.7 1859 1939 forward 2 TM 233 233003.6 402 479 forward 3 TM 236 216189.2 843 920 forward 3 SP 236 216189.2 1332 1418 forward 3 SP 236 216189.2 1035 1118 forward 3 SP 238 382906.16 155 238 forward 2 SP 241 346016.5 1841 1924 forward 2 SP 241 346016.5 1921 2013 forward 1 SP 241 346016.5 2054 2161 forward 2 SP 241 346016.5 359 448 forward 2 SP 242 346016.6 1836 1919 forward 3 SP 242 346016.6 1799 1891 forward 2 SP 242 346016.6 2049 2156 forward 3 SP 242 346016.6 1737 1829 forward 3 SP 242 346016.6 4777 4854 forward 1 SP 244 413835.5 3440 3517 forward 2 SP 246 474673.1 5054 5125 forward 2 TM 246 474673.1 6072 6155 forward 3 TM 246 474673.1 1883 1966 forward 2 TM 246 474673.1 6379 6456 forward 1 TM 246 474673.1 7446 7526 forward 3 TM 246 474673.1 8881 8964 forward 1 TM 248 402640.1 449 526 forward 2 TM 248 402640.1 369 476 forward 3 SP 252 253946.17 6747 6842 forward 3 SP 252 253946.17 4350 4430 forward 3 TM 252 253946.17 5326 5406 forward 1 TM 252 253946.17 7207 7284 forward 1 TM 252 253946.17 245 322 forward 2 SP 254 997704.1 209 322 forward 2 SP 261 82168.5 1162 1248 forward 1 SP 261 82168.5 1456 1530 forward 1 TM 261 82168.5 7638 7718 forward 3 TM 261 82168.5 7016 7099 forward 2 SP 263 412661.2 10202 10297 forward 2 SP 263 412661.2 1842 1919 forward 3 SP 264 206310.2 1856 1933 forward 2 SP 266 385608.47 3643 3720 forward 1 TM 267 385608.2 422 529 forward 2 SP 267 385608.2 260 340 forward 2 TM 267 385608.2 890 973 forward 2 TM 267 385608.2 473 550 forward 2 TM 269 978402.3 159 242 forward 3 SP 270 232146.1 1274 1387 forward 2 SP 273 239797.3 346 426 forward 1 SP 276 427554.6 59 154 forward 2 SP 276 427554.6 79 156 forward 1 TM 278 82154.23 267 347 forward 3 SP 280 1137293.16 146 226 forward 2 SP 288 477387.7 661 744 forward 1 SP 290 350440.15 1123 1203 forward 1 SP 290 350440.15 3629 3703 forward 2 SP 290 350440.15 3972 4052 forward 3 TM 290 350440.15 3146 3226 forward 2 SP 290 350440.15 3377 3460 forward 2 SP 292 997395.4 509 589 forward 2 SP 295 201436.3 1688 1771 forward 2 SP 295 201436.3 1658 1735 forward 2 TM 296 198947.1 422 502 forward 2 SP 296 198947.1 451 543 forward 1 SP 296 198947.1 231 308 forward 3 SP 304 400280.3 4930 5010 forward 1 SP 304 400280.3 766 855 forward 1 SP 304 400280.3 6645 6722 forward 3 TM 304 400280.3 134 217 forward 2 TM 304 400280.3 137 217 forward 2 SP 304 400280.3 871 972 forward 1 SP 304 400280.3 2963 3046 forward 2 SP 304 400280.3 4929 5012 forward 3 TM 304 400280.3 606 695 forward 3 TM 304 400280.3 5865 5951 forward 3 TM 304 400280.3 2746 2820 forward 1 TM 305 17320.1 1071 1151 forward 3 SP 308 107405.1 305 382 forward 2 TM 310 337024.3 1776 1859 forward 3 TM 318 481847.1 122 214 forward 2 SP 319 235636.1 426 506 forward 3 TM 322 16760.1 139 225 forward 1 SP 323 198522.1 684 764 forward 3 TM 323 198522.1 297 380 forward 3 TM 329 903269.4 1479 1562 forward 3 TM 329 903269.4 4041 4148 forward 3 SP 329 903269.4 180 266 forward 3 SP 330 351166.1 183 257 forward 3 TM 331 344713.3 168 254 forward 3 TM 332 167920.1 26 109 forward 2 TM 332 167920.1 188 271 forward 2 TM 333 433569.1 970 1044 forward 1 SP 333 433569.1 149 241 forward 2 SP 335 348121.9 4210 4299 forward 1 SP 341 405041.1 94 186 forward 1 SP 342 474266.2 2672 2749 forward 2 TM 342 474266.2 617 700 forward 2 SP 342 474266.2 2725 2808 forward 1 TM 347 372313.6 2350 2424 forward 1 TM 348 256871.2 5 100 forward 2 SP 348 256871.2 5 100 forward 2 SP 350 22485.15 308 394 forward 2 SP 350 22485.15 13 102 forward 1 SP 351 22485.1 360 446 forward 3 SP 351 22485.1 65 154 forward 2 SP 352 26410.1 337 417 forward 1 TM 354 472165.22 1863 1949 forward 3 SP 355 206580.1 227 307 forward 2 TM 358 416874.3 76 165 forward 1 SP 364 449173.16 1573 1653 forward 1 TM 367 252747.27 270 353 forward 3 SP 369 1000084.27 4622 4705 forward 2 SP 369 1000084.27 309 410 forward 3 SP 369 1000084.27 1089 1169 forward 3 SP 369 1000084.27 4170 4259 forward 3 SP 369 1000084.27 4040 4123 forward 2 SP 369 1000084.27 4138 4227 forward 1 SP 370 205607.5 2447 2533 forward 2 SP 370 205607.5 2060 2146 forward 2 SP 371 1089426.1 625 714 forward 1 SP 371 1089426.1 482 562 forward 2 SP 374 474724.5 3145 3222 forward 1 TM 374 474724.5 2538 2618 forward 3 TM 374 474724.5 3103 3189 forward 1 SP 374 474724.5 1154 1234 forward 2 TM 374 474724.5 3544 3630 forward 1 TM 377 229298.1 2450 2551 forward 2 SP 377 229298.1 923 1015 forward 2 SP 378 229298.2 28 114 forward 1 SP 378 229298.2 128 220 forward 2 SP 384 344398.2 1812 1889 forward 3 TM 384 344398.2 2095 2172 forward 1 SP 394 238342.1 1451 1534 forward 2 SP 394 238342.1 2636 2716 forward 2 SP 397 347865.4 584 664 forward 2 SP 398 40057.2 1114 1209 forward 1 SP 404 238071.2 3338 3415 forward 2 TM 404 238071.2 969 1049 forward 3 SP 404 238071.2 3274 3357 forward 1 TM 404 238071.2 593 673 forward 2 SP 404 238071.2 630 704 forward 3 TM 404 238071.2 3301 3381 forward 1 SP 407 221812.1 10 96 forward 1 TM 410 347796.7 2467 2550 forward 1 TM 411 411474.17 4552 4647 forward 1 SP 416 153860.6 454 543 forward 1 SP 417 154178.1 270 356 forward 3 SP 418 337888.3 1716 1799 forward 3 TM 418 337888.3 963 1040 forward 3 TM 420 160011.1 419 505 forward 2 TM 420 160011.1 447 530 forward 3 TM 427 1091415.2 226 315 forward 1 SP 431 464689.22 4918 4998 forward 1 TM 434 351241.1 139 219 forward 1 TM 436 482336.31 1147 1230 forward 1 SP 437 197538.2 358 465 forward 1 SP 438 197538.8 795 875 forward 3 TM 442 210945.6 513 605 forward 3 SP 442 210945.6 408 488 forward 3 SP 444 403448.2 2437 2517 forward 1 TM 444 403448.2 1994 2071 forward 2 TM 444 403448.2 2034 2114 forward 3 TM 446 146382.22 1394 1486 forward 2 SP 447 146382.25 36 128 forward 3 SP 448 345705.7 365 448 forward 2 SP 448 345705.7 101 190 forward 2 SP 452 900031.4 2733 2816 forward 3 SP 452 900031.4 4841 4939 forward 2 SP 452 900031.4 4494 4574 forward 3 SP 453 475365.4 1519 1599 forward 1 SP 454 222810.1 161 244 forward 2 TM 455 1084493.6 2952 3044 forward 3 SP 456 979005.2 1477 1557 forward 1 TM 456 979005.2 2740 2820 forward 1 TM 456 979005.2 1409 1486 forward 2 TM 457 346686.23 2997 3080 forward 3 SP 457 346686.23 1993 2076 forward 1 SP 457 346686.23 2184 2258 forward 3 TM 457 346686.23 303 389 forward 3 SP 457 346686.23 2151 2240 forward 3 SP 458 234568.25 202 285 forward 1 SP 459 234568.17 1841 1933 forward 2 SP 459 234568.17 2059 2136 forward 1 TM 459 234568.17 208 291 forward 1 SP 462 243103.24 164 241 forward 2 SP 462 243103.24 588 674 forward 3 SP 463 469883.1 2657 2734 forward 2 SP 463 469883.1 4492 4572 forward 1 SP 463 469883.1 4587 4670 forward 3 SP 463 469883.1 1198 1287 forward 1 SP 463 469883.1 1732 1821 forward 1 SP 463 469883.1 4352 4435 forward 2 TM 463 469883.1 3980 4060 forward 2 SP 463 469883.1 2724 2813 forward 3 SP 463 469883.1 400 486 forward 1 SP 465 227932.2 4290 4370 forward 3 TM 465 227932.2 3078 3155 forward 3 TM 466 903691.6 195 278 forward 3 SP 466 903691.6 2840 2929 forward 2 SP 468 407938.5 3676 3756 forward 1 TM 469 233778.1 13 93 forward 1 SP 469 233778.1 371 451 forward 2 SP 470 474630.19 2277 2369 forward 3 SP 470 474630.19 156 236 forward 3 SP 474 228860.6 1009 1098 forward 1 SP 477 347005.5 755 844 forward 2 SP 477 347005.5 524 607 forward 2 TM 477 347005.5 1931 2017 forward 2 SP 477 347005.5 4970 5053 forward 2 SP 480 252570.6c 215 295 forward 2 SP 481 466402.116 1085 1177 forward 2 SP 483 899612.2 393 479 forward 3 SP 484 254107.1 1087 1170 forward 1 SP 484 254107.1 2929 3054 forward 1 SP 484 254107.1 3266 3349 forward 2 SP 485 208328.1 2434 2517 forward 1 SP 485 208328.1 2158 2244 forward 1 SP 486 130157.2 1344 1421 forward 3 TM 486 130157.2 1994 2074 forward 2 TM 486 130157.2 2041 2121 forward 1 TM 488 233089.1 1707 1787 forward 3 TM 489 336256.1 2057 2140 forward 2 SP 489 336256.1 1835 1918 forward 2 SP 489 336256.1 3340 3420 forward 1 TM 489 336256.1 236 319 forward 2 SP 490 979616.2 319 393 forward 1 TM 491 351432.3 4887 4964 forward 3 SP 494 481251.3 1966 2043 forward 1 TM 494 481251.3 1694 1774 forward 2 SP 494 481251.3 1825 1908 forward 1 TM 496 96700.1 141 218 forward 3 TM 499 344582.19 161 247 forward 2 SP
[0180] 4 TABLE 4 SEQ ID NO TEMPLATE ID CLONE ID START STOP 1 441269.2 467767H1 1083 1317 1 441269.2 467767R6 1083 1491 1 441269.2 467767T6 1523 2042 2 3161.5 897743R6 2385 2856 2 3161.5 897743H1 2385 2549 3 977955.7 908504T6 3163 3619 3 977955.7 908504H1 549 748 3 977955.7 908504R6 549 947 4 350754.2 981592R6 1400 1980 4 350754.2 981592H1 1400 1658 4 350754.2 981592T6 2173 2701 5 235191.4 1308478H1 568 829 5 235191.4 1308478F6 568 829 5 235191.4 1308478T6 570 791 5 235191.4 1308478R1 656 844 6 1099593.13 1435427F6 720 1228 6 1099593.13 1435427H1 720 988 6 1099593.13 1435427F1 720 1227 7 1099593.2 1435427T6 692 977 8 003161.7c 1484773F6 270 603 8 003161.7c 1484773T6 271 578 8 003161.7c 1484773H1 270 468 9 415650.5 1485735F6 80 588 9 415650.5 1485735H1 80 337 10 204401.1 1485735T6 8 579 11 347915.14 1503076T6 4750 5381 11 347915.14 1503076H1 4558 4847 11 347915.14 1503076F6 4558 5053 12 997395.1 1559836T6 2134 2777 12 997395.1 1559836H1 1946 2154 12 997395.1 1559836F6 1946 2323 13 332783.1 2450712F6 1161 1558 13 332783.1 2450712H1 1161 1400 13 332783.1 1612714F6 1653 2118 13 332783.1 1612714H1 1653 1866 13 332783.1 1612714T6 2047 2587 13 332783.1 2450712T6 2156 2648 14 422289.1 1676442H1 1178 1414 14 422289.1 1676442F6 1178 1661 14 422289.1 1676442T6 2168 2501 15 899410.5 1724967F6 920 1312 15 899410.5 1724967H1 1080 1312 16 404155.2 1724967T6 91 570 17 412065.22 2591494T6 1041 1681 17 412065.22 2591494F6 763 1349 17 412065.22 1737926F6 530 663 17 412065.22 1737926T6 1241 1679 18 412065.2 1737926H1 1 217 19 345860.21 1749417H1 309 624 19 345860.21 1749417F6 309 772 19 345860.21 1749417T6 1348 1440 20 199101.1 1758055H1 1553 1823 20 199101.1 1758055R6 1553 2065 20 199101.1 1758055T6 2470 2920 21 196606.8c 1806542H1 2386 2681 21 196606.8c 1806542F6 2116 2681 21 196606.8c 1806542T6 289 780 22 475146.3 1807407T6 1489 1940 22 475146.3 1807407F6 466 1006 22 475146.3 1807407H1 466 714 23 474069.8 1811369H1 844 1150 23 474069.8 1811369F6 844 1283 23 474069.8 1811369T6 1722 2152 24 255115.2 1846973H1 865 1117 25 255115.4 1846973T6 1381 1829 26 216331.1 1869130H1 1226 1502 26 216331.1 1869130F6 1226 1653 26 216331.1 1869130T6 1406 1976 27 482517.3 1880840H1 852 999 27 482517.3 1880840F6 852 1289 27 482517.3 1880840T6 1593 2091 28 480653.1 1901284F6 111 569 28 480653.1 1901284H1 111 367 28 480653.1 1901284T6 666 1201 29 978047.1 1922875T6 1398 1987 29 978047.1 1922875H1 846 1029 29 978047.1 1922875R6 847 1347 30 330977.1c 1998075H1 821 1095 30 330977.1c 1998075R6 706 1095 30 330977.1c 1998075T6 676 1068 31 343502.9 1998428H1 617 889 31 343502.9 1998428R6 617 1062 31 343502.9 1998428T6 617 974 32 903104.11c 2054053R6 514 1051 32 903104.11c 2054053H1 774 1051 33 898068.6 2054053T6 748 1276 34 903104.1 2263572R6 112 583 34 903104.1 2263572H1 95 346 35 903104.8 2054053CB1 2 2370 35 903104.8 2263572T6 1786 2241 36 412065.21 2591494H1 1010 1179 37 399067.1 2615184CA2 1 2855 37 399067.1 2615184H1 1 251 37 399067.1 2615184F6 1 380 37 399067.1 2615184T6 2108 2557 38 237709.11 2625371F6 1145 1704 38 237709.11 2625371T6 1166 1788 39 237709.5 2625371H1 1732 2007 40 216437.4 2636043CB1 98 1101 40 216437.4 2636043H1 998 1085 40 216437.4 2636043F6 492 1085 41 255824.52 2636043T6 1552 1836 42 27798.1 2653855T6 3882 4203 42 27798.1 2653855H1 2809 3102 42 27798.1 2653855F6 2809 3399 43 60671.7 2676441F6 584 989 43 60671.7 2676441H1 584 829 43 60671.7 2582525T6 1814 2355 44 376085.9 2676441T6 1998 2206 45 404467.1 2715440H1 1 242 45 404467.1 2715440F6 1 530 46 903508.12 2715440T6 2750 2967 47 334387.1 2718810T6 2277 2764 47 334387.1 2718810CB1 1 2834 47 334387.1 2718810F6 1239 1742 47 334387.1 2718810H1 1239 1496 48 290403.5 2839245F6 1055 1548 48 290403.5 2839245H1 1055 1319 49 391406.5 2883195CB1 1848 2974 49 391406.5 2883195F6 1912 2425 50 391406.24 2883195H1 4 277 50 391406.24 2883195T6 330 925 51 474588.21 3070625H1 1762 2041 51 474588.21 3070625F6 1761 2181 52 124541.1 3176339F6 3891 4412 52 124541.1 3176339T6 8 409 52 124541.1 3176339H1 3894 4135 53 246862.9 3206210F6 46 466 53 246862.9 3206210H1 46 149 54 246862.17 3206210T6 1519 1947 55 13040.1 3436305F6 106 357 55 13040.1 3436305H1 106 219 56 236480.3 3436305T6 3031 3443 57 314831.5 135769H1 1362 1577 57 314831.5 135769R6 1362 1874 57 314831.5 135769T6 1984 2532 58 391940.1 343763R6 2988 3443 58 391940.1 343763H1 2989 3091 58 391940.1 343763T6 3132 3646 59 454958.13 591358R1 582 929 59 454958.13 591358H1 582 729 59 454958.13 591358R6 582 938 60 1090481.2c 591358T6 1 252 61 238203.11 999864R6 4229 4728 61 238203.11 999864H1 4229 4313 61 238203.11 999864T6 4548 5093 62 994057.21 1749663H1 1576 1716 62 994057.21 1749663F6 1576 2017 63 346730.5 1818365H1 548 841 63 346730.5 1818365F6 548 1047 64 346730.2c 1818365T6 1 563 65 458903.1 1962141H1 353 588 66 482336.2 1962141R6 907 1438 67 482336.14 1962141T6 1320 1926 68 66522.1 1965083H1 1 266 68 66522.1 1965083R6 1 410 69 300294.3 1984158H1 2131 2393 69 300294.3 1984158R6 2131 2454 69 300294.3 1984158T6 2131 2429 70 474372.8 1988774R6 2510 2848 70 474372.8 1988774H1 2510 2780 71 475028.7 2057432CB1 1 2100 71 475028.7 2057432H1 140 427 71 475028.7 2057432R6 140 704 72 481223.3 2057432T6 2099 2683 73 332499.1 2123889T6 2129 2313 73 332499.1 2123889F6 2129 2350 73 332499.1 2123889H1 2129 2350 74 234340.7 2189132F6 17 458 74 234340.7 2189132H1 17 300 75 234340.15c 2189132T6 60 513 76 255778.11 2374121H1 438 632 76 255778.11 2374121T6 1352 1841 76 255778.11 2374121CB1 1 958 76 255778.11 2374121F6 438 958 76 255778.11 2374121CA2 438 958 77 317586.1 2613155T6 1232 1666 77 317586.1 2613155F6 1513 1705 77 317586.1 2613155H1 1531 1705 78 1093574.1c 2648611F6 607 1089 78 1093574.1c 2648611H1 607 841 79 355658.1 2648611T6 1 233 80 201342.4 2802809T6 2155 2700 80 201342.4 2802809H1 1365 1634 80 201342.4 2802809F6 1365 1793 81 204542.1 2817637H1 2 266 82 428742.1 2817637T6 1 339 83 204386.1 3296181T6 2265 2823 83 204386.1 3296181H1 293 493 83 204386.1 3296181F6 293 698 84 335086.1 3602403H1 3018 3210 84 335086.1 3602403F6 3018 3387 85 228302.1 3602403T6 634 706 86 1063057.1 4140129T6 192 718 86 1063057.1 4140129H1 1 295 86 1063057.1 4140129F6 1 450 87 199905.1 384911H1 1883 2162 87 199905.1 384911R6 1888 2281 88 11329.1 501563T6 404 670 88 11329.1 501563H1 404 563 88 11329.1 501563R6 404 707 89 237536.18 549297H1 735 1047 89 237536.18 549297R6 766 1047 89 237536.18 549297T6 296 785 90 330878.6 642320R6 1556 2014 90 330878.6 642320H1 1556 1813 90 330878.6 642320T6 3156 3745 91 331793.11 737335CA2 112 1166 91 331793.11 737335H1 112 339 91 331793.11 737335R6 112 470 91 331793.11 737335T6 549 1116 92 902895.2 1234126F1 409 724 92 902895.2 1234126F6 409 724 92 902895.2 1234126H1 409 660 92 902895.2 1234126T6 409 674 93 7273.1 1367516R1 91 519 93 7273.1 1367516H1 91 228 93 7273.1 1367516R6 91 518 93 7273.1 1367516T6 171 565 94 190771.2 1394614F6 729 1173 94 190771.2 1394614H1 729 996 94 190771.2 1394614T6 1986 2563 95 1095702.14 1428779CB1 94 2366 95 1095702.14 1428779H1 111 360 95 1095702.14 1428779F6 111 406 96 1095702.4c 1428779T6 5160 5336 97 977667.1 1446659H1 537 786 97 977667.1 1446659F6 537 979 97 977667.1 1446659T6 32 96 98 253534.14 1450849T6 1 417 98 253534.14 1450849H1 1 135 98 253534.14 1450849F6 1 443 98 253534.14 1450849H6 1 218 99 1136709.5 1488704H1 1 199 99 1136709.5 1488704CA2 1 768 99 1136709.5 1488704F6 1 511 100 1136709.6 1488704T6 116 578 101 382293.16 1599046H1 258 475 101 382293.16 1599046F6 258 655 102 382293.15 1599046T6 2489 2898 103 235943.39c 1603809F6 2213 2678 103 235943.39c 1603809CA2 1307 2678 104 235943.36 1603809H1 4 224 105 401434.1 1610682T6 1024 1310 105 401434.1 1610682H1 1031 1225 105 401434.1 1610682F6 1031 1351 106 234107.2 1615959T6 1788 2370 106 234107.2 1615959H1 695 885 106 234107.2 1615959F6 695 1056 107 233660.2c 1626152F6 4392 4768 107 233660.2c 1626152H1 4558 4768 107 233660.2c 1626152T6 2831 3411 108 482423.1 1648841H1 696 856 108 482423.1 1648841F6 696 1033 109 441298.14 1676389T6 4775 5183 109 441298.14 1676389H1 3121 3333 109 441298.14 1676389F6 3121 3619 110 235333.1 1684632H1 739 945 110 235333.1 1684632F6 739 1215 110 235333.1 1684632T6 835 1320 111 221872.11c 1722234F6 799 1068 111 221872.11c 1722234H1 860 1068 112 992317.12 1725322T6 864 1342 112 992317.12 1725322F6 866 1269 112 992317.12 1725322H1 866 1082 113 230936.6 1747818T6 1813 2350 113 230936.6 1747818F6 2170 2729 113 230936.6 1747818H1 2462 2729 114 029508.1c 1798468F6 97 539 114 029508.1c 1798468H1 99 370 115 984900.1 1802185H1 29 180 115 984900.1 1802185F6 29 322 116 405158.1 1806433F6 1972 2176 116 405158.1 1806433H1 2019 2240 116 405158.1 1806433T6 2785 3209 117 334177.1 1849820H1 978 1232 117 334177.1 1849820F6 978 1434 118 480187.81 1870759CB1 1 1386 118 480187.81 1870759F6 36 467 118 480187.81 1870759H1 36 299 119 62042.4 1979528H1 1040 1286 119 62042.4 1979528R6 1040 1419 119 62042.4 1979528T6 1075 1534 120 1087696.7 2076621H1 10 273 120 1087696.7 2076621F6 10 428 121 259805.28 2076621T6 2555 2881 122 399128.1 2097461H1 1 225 122 399128.1 2097461R6 2 483 123 986565.15 2191455F6 603 936 123 986565.15 2191455H1 603 853 123 986565.15 2191455T6 3044 3532 124 978378.3 2202952H1 700 934 124 978378.3 2202952F6 700 1002 125 979575.2c 2243954F6 672 1113 125 979575.2c 2243954H1 862 1113 126 233594.4 2264081R6 61 348 126 233594.4 2264081H1 61 317 127 238635.1 2365066H1 232 464 127 238635.1 2365066F6 232 774 127 238635.1 2365066T6 466 882 128 1080496.1 2414663F6 599 1131 128 1080496.1 2414663H1 599 823 129 351204.2 2414663T6 702 990 130 235394.5 2447071F6 2469 2875 130 235394.5 2447071H1 2469 2686 131 403242.1 2500915F6 1724 2064 131 403242.1 2500915H1 1724 1949 131 403242.1 2500915T6 1883 2304 132 427529.9 2504404F6 22 511 132 427529.9 2504404H1 22 262 133 474868.2 2525691T6 1904 2376 133 474868.2 2525691F6 95 512 133 474868.2 2525691H1 95 360 133 474868.2 2525691CA2 100 2457 133 474868.2 2525691CB1 102 2450 134 1095839.17 2541141F6 44 531 134 1095839.17 2541141H1 44 276 135 1095839.1 2541141T6 3113 3589 136 244251.4 2586674T6 543 974 136 244251.4 2586674H1 1 265 136 244251.4 2586674F6 1 308 137 21555.1 2611420H1 1 251 137 21555.1 2611420F6 1 373 138 234157.3 2621425H1 939 1200 138 234157.3 2621425R6 939 1376 139 903478.1 2621425T6 1 559 140 411296.2 2651610T6 1667 2272 140 411296.2 2651610F6 1326 1846 140 411296.2 2651610H1 1293 1540 141 18044.1 2652691F6 93 574 141 18044.1 2652691H1 93 330 142 407260.2 2669708F6 768 1327 142 407260.2 2669708H1 768 1019 142 407260.2 2669708T6 906 1483 143 978765.2 2756450R6 1 194 143 978765.2 2756450T6 1 156 144 338274.1 2830040F6 8 458 144 338274.1 2830040T6 1 507 144 338274.1 2830040H1 8 265 145 231270.1 2905430H1 606 678 145 231270.1 2905430F6 606 938 146 18092.1 2905612H1 1 190 146 18092.1 2905612F6 1 190 147 404197.8c 2964015F6 16 413 147 404197.8c 2964015H1 103 413 148 404197.4 2964015T6 2687 3114 149 5460.1 3124174T6 87 566 149 5460.1 3124174F6 7 349 149 5460.1 3124174H1 7 292 150 346511.4 3214930F6 81 650 151 346511.6 3214930H2 17 125 152 331233.2 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165 100653.3 025818H1 431 632 166 978478.4 041692H1 494 741 166 978478.4 041692R6 494 831 167 85942.2 109658R6 44 521 168 85942.3 109658T6 416 986 169 977470.16c 155890T6 64 477 169 977470.16c 155890H1 1926 2123 169 977470.16c 155890R6 1785 2123 170 283762.2 156350R6 1538 1813 170 283762.2 156350H1 1538 1713 171 331065.2 162793H1 1798 2217 172 331065.5 162793R6 1666 2059 173 252782.4 162793T6 2178 2789 174 229068.1 197779R6 359 821 174 229068.1 197779H1 359 584 174 229068.1 197779T6 745 1317 175 407450.7 275683R6 188 447 175 407450.7 275683H1 188 432 175 407450.7 275683T6 280 877 176 200163.2 348429R6 437 979 176 200163.2 348429R1 437 917 176 200163.2 348429F1 915 1497 176 200163.2 348429T7 948 1459 176 200163.2 348429T6 1248 1454 177 200163.12c 348429CB1 7 1801 177 200163.12c 348429H1 171 401 178 1328237.4 452790T7 2338 2685 178 1328237.4 452790R6 976 1423 178 1328237.4 452790H1 976 1211 178 1328237.4 452790R7 976 1406 179 333965.1 452790T6 1905 2426 180 1328236.5 515029H1 847 1071 180 1328236.5 515029R6 597 1070 180 1328236.5 515029T6 169 776 181 995534.3 546656H1 1514 1803 181 995534.3 546656R6 1514 1973 181 995534.3 546656T6 2012 2433 181 995534.3 546656CB1 70 4457 182 1098887.1 569710R1 191 728 182 1098887.1 2317772T6 922 1262 182 1098887.1 569710H1 190 439 182 1098887.1 569710R6 190 729 182 1098887.1 569710CB1 1 1309 182 1098887.1 569710CA2 191 1323 182 1098887.1 2317772H1 511 765 182 1098887.1 2317772R6 511 987 182 1098887.1 569710F1 691 1309 182 1098887.1 569710T6 813 1271 183 268733.1 586337H1 1 239 183 268733.1 586337R6 1 239 184 470023.2 610280R6 292 808 184 470023.2 610280H1 292 563 185 989966.8 610280T6 1955 2086 186 233925.5 637639R6 833 1195 186 233925.5 637639H1 833 1089 186 233925.5 637639T6 1079 1694 187 405145.5 855379R1 7249 7460 187 405145.5 855379R6 7249 7460 187 405145.5 855379H1 7249 7460 188 261982.8 1234054CB1 698 8277 188 261982.8 4106629F6 992 1484 188 261982.8 4106629H1 994 1275 188 261982.8 4106629T6 2457 2747 188 261982.8 1234054H1 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402187.16 1607765H1 1033 1239 213 402187.16 1607765T6 1047 1370 214 903091.31 1618369CA2 145 1709 215 903091.33c 1618369F6 2363 2905 215 903091.33c 1618369CB1 754 2904 216 903091.16 1618369H1 1 219 217 228447.29c 1639231F6 1 230 217 228447.29c 1639231H1 1 201 218 475473.1 1649906H1 1155 1354 218 475473.1 1649906F6 1155 1543 219 354430.4 1661486F6 3990 4349 219 354430.4 1661486H1 3990 4201 219 354430.4 1661486T6 6224 6432 220 468221.12c 1662856F6 257 765 220 468221.12c 1662856H1 257 484 221 468221.13 1662856T6 992 1313 222 399187.1c 1669720F6 1 398 222 399187.1c 1669720H1 300 398 223 903338.12 1669720T6 157 508 224 208529.1 1676657T6 720 1170 224 208529.1 1676657F6 524 900 224 208529.1 1676657H1 524 767 225 1040667.52 1684010H1 482 696 225 1040667.52 1684010F6 482 962 226 147665.1 1709754H1 492 706 226 147665.1 1709754F6 492 787 226 147665.1 1709754T6 492 749 227 218524.4 1715437F6 1103 1570 227 218524.4 1715437H1 1103 1373 227 218524.4 1715437T6 1352 1767 228 346673.1 1724856F6 2244 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2172334F6 1163 1711 252 253946.17 2172334H1 1163 1418 252 253946.17 2172334T6 1891 2457 253 13550.1 2190842CB1 63 584 253 13550.1 2190842F6 155 579 253 13550.1 2190842H1 155 325 253 13550.1 2190842T6 207 537 254 997704.1 2191181H1 856 1086 254 997704.1 2191181F6 856 1089 254 997704.1 2191181T6 860 1044 255 385608.45 2219234F6 878 1327 256 385608.25c 2219234H1 682 935 257 385608.3 2219234T6 3156 3676 258 78434.1 2308788T6 1 141 258 78434.1 2308788R6 1 183 258 78434.1 2308788H1 1 193 259 243134.1 2355979T6 5 298 259 243134.1 2355979F6 9 304 259 243134.1 2355979H1 80 304 260 196959.4c 2358377CB1 1222 3902 260 196959.4c 2358377R6 3476 3902 260 196959.4c 2358377H1 3676 3902 261 82168.5 2450092F6 1 441 261 82168.5 2450092H1 1 221 262 4360.1 2458924F6 172 614 262 4360.1 2458924H1 172 402 262 4360.1 2458924T6 549 951 263 412661.2 2470485F6 481 1054 263 412661.2 2470485H1 481 732 263 412661.2 172023R6 7002 7403 263 412661.2 172023H1 7002 7190 264 206310.2 2486065H1 1747 1973 264 206310.2 2486065F6 1747 1836 265 346599.14c 2515389F6 726 1170 265 346599.14c 2515389H1 845 1170 266 385608.47 2520418F6 309 681 267 385608.2 2520418CB1 1340 4480 267 385608.2 2520418H1 1330 1618 268 335202.1 2530672T6 438 549 268 335202.1 2530672F6 445 719 268 335202.1 2530672H1 445 702 269 978402.3 2553094H1 263 505 269 978402.3 2553094F6 263 605 270 232146.1 2578906T6 2675 2967 270 232146.1 2578906H1 381 624 270 232146.1 2578906F6 381 798 271 25757.1 2588149F6 110 371 271 25757.1 2588149H1 110 340 271 25757.1 2588149T6 110 329 272 206860.2 2589404F6 1 199 272 206860.2 2589404H1 1 199 272 206860.2 2589404T6 25 199 273 239797.3 2598317F6 656 1017 273 239797.3 2598317H1 656 903 273 239797.3 2598317T6 664 984 274 441328.12 2601777F6 2979 3491 274 441328.12 2601777H1 2979 3262 274 441328.12 2601777T6 2972 3454 275 113975.1 2628789F6 1 318 275 113975.1 2628789H1 1 229 275 113975.1 2628789T6 179 507 276 427554.6 2646362CB1 232 936 276 427554.6 2646362F6 284 674 276 427554.6 2646362CA2 284 754 276 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408116.1 4190635T6 2 145 338 339737.1c 4252048F6 1 384 338 339737.1c 4252048H1 146 384 339 978146.1 4416082H1 436 673 339 978146.1 4416082F6 436 735 339 978146.1 4416082T6 512 735 340 186012.1 4641887F6 410 697 340 186012.1 4641887H1 411 639 341 405041.1 4754771F6 411 855 341 405041.1 4754771H1 411 679 342 474266.2 4754771T6 1928 2239 343 401530.2 4765137H1 384 643 343 401530.2 4765137F6 385 685 344 234729.11 4765137T6 433 607 345 195199.1 4936477F6 1178 1498 345 195199.1 4936477H1 1179 1394 345 195199.1 4936477T6 1822 2073 346 368731.1 5098311T6 71 515 346 368731.1 5098311F6 1 499 346 368731.1 5098311H1 1 262 347 372313.6 5260618F6 172 611 347 372313.6 5260618H1 172 409 347 372313.6 5260618T6 387 875 348 256871.2 5675077H1 169 286 348 256871.2 5675077F6 169 549 349 256871.16c 5675077T6 219 640 350 22485.15 342285H1 305 537 351 22485.1 342285R6 357 859 351 22485.1 342285CB1 6 1038 352 26410.1 443631H1 44 285 352 26410.1 443631R6 44 453 352 26410.1 443631T6 186 521 353 215720.1 630607R6 1 389 353 215720.1 630607H1 1 148 354 472165.22 827257R6 1 338 354 472165.22 827257T1 2 338 354 472165.22 827257T6 2 284 354 472165.22 827257R1 2 338 354 472165.22 827257H1 1 284 355 206580.1 919907H1 136 438 355 206580.1 919907R6 136 615 355 206580.1 919907T6 204 629 356 344775.3 1292238F6 1929 2096 356 344775.3 1292238H1 1929 2095 356 344775.3 1292238T6 2155 2495 357 334668.1 1417114F6 797 956 357 334668.1 1417114H1 797 956 357 334668.1 1417114T6 797 916 358 416874.3 1445547H1 1416 1678 358 416874.3 1445547F6 1416 1949 358 416874.3 1445547T6 1677 2292 359 427964.2 1507768T6 1138 1508 359 427964.2 1507768F6 1145 1548 359 427964.2 1507768H1 1145 1345 360 234537.3c 1718651H1 421 608 360 234537.3c 1718651F6 150 608 360 234537.3c 1718651T6 31 571 360 234537.3c 2169651T6 44 556 360 234537.3c 2169651F6 2789 3156 360 234537.3c 2169651H1 2915 3156 361 31760.1 1740072R6 1 339 361 31760.1 1740072H1 1 242 361 31760.1 1740072T6 1 302 362 336953.7 1857708H1 5175 5437 362 336953.7 1857708F6 5175 5635 362 336953.7 1857708T6 5518 5964 363 444771.2 2061119H1 321 590 363 444771.2 2061119R6 321 619 363 444771.2 2061119T6 321 585 364 449173.16 2062092R6 2140 2505 364 449173.16 2062092H1 2140 2359 365 1523.1 2176222F6 1 481 365 1523.1 2176222H1 1 246 366 239097.1 2176222T6 15 403 367 252747.27 2829958H1 865 1078 367 252747.27 2829958F6 865 1242 368 482490.11 2888737F6 1 255 368 482490.11 2888737H1 5 255 368 482490.11 2888737T6 39 262 369 1000084.27 3679667F6 1805 2138 369 1000084.27 3679667H1 1986 2138 369 1000084.27 3679667T6 1646 2012 370 205607.5 009140H1 1915 2180 370 205607.5 009140CB1 1 3018 371 1089426.1 041762H1 1 82 372 335186.2 056197H1 351 548 373 357276.8 391601R6 1 449 373 357276.8 391601T6 122 594 374 474724.5 392166T6 3735 4307 374 474724.5 392166R6 3753 4182 375 230889.3 413767H1 790 1010 375 230889.3 413767R6 790 1227 375 230889.3 413767T6 1467 2064 376 903909.1 660142T6 1168 1598 376 903909.1 660142H1 778 1006 376 903909.1 660142R6 784 999 377 229298.1 1320006T6 2658 3178 377 229298.1 1320006F6 2510 2966 378 229298.2 1320006H1 1690 1930 379 110678.1 1381722H1 823 920 379 110678.1 1381722F6 823 920 379 110678.1 1381722T6 825 920 380 239093.1 1506104T6 1 466 380 239093.1 1506104F6 8 442 380 239093.1 1506104H1 8 204 381 237963.11c 1561444F6 458 805 381 237963.11c 1561444H1 603 805 382 237963.8 1561444T6 1409 1797 383 400135.1 1562305H1 1 216 383 400135.1 1562305F6 1 538 384 344398.2 1575439T6 1733 2313 384 344398.2 1575439F6 2082 2454 384 344398.2 1575439H1 2223 2454 385 1086647.1 1676601T6 632 760 385 1086647.1 1676601F6 633 760 385 1086647.1 1676601H1 633 760 386 1682.1 1676627T6 258 635 386 1682.1 1676627F6 265 700 386 1682.1 1676627H1 265 481 387 010190.1c 1705809F6 62 330 387 010190.1c 1705809H1 113 330 387 010190.1c 1705809T6 62 312 388 205311.1 1717242F6 1 355 388 205311.1 1717242H1 1 241 388 205311.1 1717242T6 40 355 389 22429.7 1726945F6 663 1163 389 22429.7 1726945H1 663 886 389 22429.7 1726945T6 931 1340 390 59379.1 1728196T6 1 190 390 59379.1 1728196F6 8 190 390 59379.1 1728196H1 8 198 391 236253.1 1812687H1 1 232 391 236253.1 1812687F6 1 422 391 236253.1 1812687T6 175 575 392 331033.1 1970111CB1 34 2891 392 331033.1 1970111F6 1092 1608 392 331033.1 1970111H1 1092 1378 392 331033.1 1970111T6 2392 2838 393 345533.8c 2052607F6 349 451 393 345533.8c 2052607H1 348 451 393 345533.8c 2052607T6 390 451 394 238342.1 2173282T6 2531 2999 394 238342.1 2173282H1 2557 2790 394 238342.1 2173282F6 2557 2957 395 28936.1 2378570F6 80 580 395 28936.1 2378570H1 80 305 396 347865.5 2470377F6 1 307 396 347865.5 2470377H1 7 105 397 347865.4 2470377T6 347 450 398 40057.2 2555937F6 487 1001 398 40057.2 2555937T6 502 719 398 40057.2 2555937H1 764 1001 399 997301.6 2665985H1 1 217 399 997301.6 2665985F6 1 489 399 997301.6 2665985T6 275 803 400 12235.1 2728985F6 1 428 400 12235.1 2728985H1 1 193 400 12235.1 2728985T6 1 403 401 245496.7 2750145H1 253 517 401 245496.7 2750145R6 253 814 401 245496.7 2750145F6 365 862 402 983262.3c 2778621F6 203 574 402 983262.3c 2778621H1 203 456 402 983262.3c 2778621T6 335 886 403 998084.1 2821452H1 34 347 403 998084.1 2821452F6 34 541 403 998084.1 2821452T6 171 741 404 238071.2 2823835T6 3140 3578 404 238071.2 2823835H1 2541 2820 404 238071.2 2823835F6 2542 3036 405 103930.1 2903975H1 35 272 405 103930.1 2903975F6 36 585 405 103930.1 2903975T6 434 670 406 254068.1 2914716F6 1 438 406 254068.1 2914716H1 2 192 406 254068.1 2914716T6 25 437 407 221812.1 2952169F6 396 768 407 221812.1 2952169H1 396 643 407 221812.1 2952169T6 388 986 408 240129.1 2956319F6 257 421 408 240129.1 2956319H1 257 421 409 230297.1c 2958047F6 1 496 409 230297.1c 2958047T6 2 453 409 230297.1c 2958047H1 1 267 410 347796.7 3108506H1 395 686 410 347796.7 3108506F6 436 687 411 411474.17 3143449R6 4838 5336 411 411474.17 3143449H1 5028 5335 412 027434.1c 3168342H1 1 145 412 027434.1c 3168342F6 3 145 413 979488.1c 3168342T6 392 565 414 213988.1 3624062F6 9 242 414 213988.1 3624062H1 11 218 414 213988.1 3624062T6 9 198 415 263336.62 279898T6 239 581 415 263336.62 279898R7 261 614 415 263336.62 279898H1 261 568 415 263336.62 279898R6 261 608 416 153860.6 645136T6 222 609 416 153860.6 645136R6 1 419 416 153860.6 645136H1 1 247 417 154178.1 666098T6 1 398 417 154178.1 666098R6 1 295 417 154178.1 666098H1 1 201 418 337888.3 668460H1 1357 1560 418 337888.3 668460R6 1357 1538 418 337888.3 668460T6 1655 1872 419 240009.2c 797955R6 1 398 419 240009.2c 797955T6 1 388 419 240009.2c 797955H1 1 123 420 160011.1 915572H1 1 225 420 160011.1 915572R6 1 393 421 332919.4 924319R6 781 1262 421 332919.4 924319H1 781 1142 422 6233.1 1357993T6 5 217 422 6233.1 1357993F6 8 258 422 6233.1 1357993H1 8 214 423 245532.17c 1738288F6 475 691 423 245532.17c 1738288H1 471 691 423 245532.17c 1738288T6 139 579 424 205486.1 1743453R6 1 397 424 205486.1 1743453H1 1 196 424 205486.1 1743453T6 324 608 425 987927.13 1798209F6 677 1233 425 987927.13 1798209H1 677 930 425 987927.13 1798209T6 2136 2698 426 25423.3 1816768H1 19 194 426 25423.3 1816768F6 19 457 426 25423.3 1816768T6 229 726 427 1091415.2 2056584R6 1217 1675 427 1091415.2 2056584H1 1217 1455 428 1091415.16c 2056584T6 178 738 429 230058.2 2500717H1 1707 1952 429 230058.2 2500717F6 1707 2165 429 230058.2 2500717T6 1939 2519 430 444648.9 2757583H1 328 585 430 444648.9 2757583R6 328 698 430 444648.9 2757583F6 328 698 430 444648.9 2757583CB1 268 709 431 464689.22 2845102F6 1659 2267 431 464689.22 2845102H1 1659 1900 432 464689.15 2845102T6 1773 2251 433 334809.3c 2935554H1 1058 1331 433 334809.3c 2935554F6 831 1331 433 334809.3c 2935554T6 38 598 434 351241.1 2955163H1 312 587 434 351241.1 2955163F6 326 757 434 351241.1 2955163T6 346 716 435 482336.11 5164266H1 1 205 435 482336.11 5164266F6 1 71 436 482336.31 5164266T6 1237 1458 437 197538.2 440472R6 585 945 437 197538.2 440472H1 585 709 438 197538.8 440472T6 642 1050 439 232048.14 548808H1 94 327 439 232048.14 548808R6 94 632 439 232048.14 548808T6 216 595 440 1092381.1 1285892H1 5090 5310 441 903804.1 1287264F6 318 575 441 903804.1 1287264H1 336 575 441 903804.1 1287264F1 394 575 442 210945.6 1288118H1 299 550 442 210945.6 1288118F6 299 688 443 210945.3c 1288118CB1 1 1120 443 210945.3c 1288118T6 539 856 444 403448.2 1292773T6 2224 2602 444 403448.2 1292773F6 2256 2647 444 403448.2 1292773H1 2269 2509 445 238263.2 1301770F6 669 1096 445 238263.2 1301770H1 669 913 445 238263.2 1301770T6 1245 1774 446 146382.22 1313615F6 1107 1702 446 146382.22 1313615H1 1107 1339 447 146382.25 1313615T6 165 467 448 345705.7 1357056H1 58 304 448 345705.7 1357056F1 58 490 448 345705.7 1357056T6 2026 2525 448 345705.7 1357056F6 58 509 448 345705.7 1357056CA2 58 2578 449 198212.1 1377880F6 262 706 449 198212.1 1377880H1 262 514 450 198212.6 1377880T6 1232 1600 451 900031.8 1402833F6 1 574 451 900031.8 1402833H1 1 233 452 900031.4 1402833T6 5643 6007 453 475365.4 1430483F6 443 829 453 475365.4 1430483H1 443 695 453 475365.4 1430483T6 1273 1699 454 222810.1 1438102T6 886 1263 454 222810.1 1438102F6 1 459 454 222810.1 1438102H1 1 263 455 1084493.6 1503350H1 1299 1571 455 1084493.6 1503350F6 1308 1629 455 1084493.6 1503350T6 2679 3224 456 979005.2 1512017H1 2127 2297 456 979005.2 1512017T6 3743 4252 456 979005.2 1512017F6 2127 2625 457 346686.23 1596060T6 2552 3060 457 346686.23 1596060H1 2040 2231 457 346686.23 1596060F6 2040 2477 458 234568.25 1624988H1 33 108 458 234568.25 1624988F6 33 458 459 234568.17 1624988T6 2421 3023 460 978276.1c 1639657H1 387 546 460 978276.1c 1639657F6 278 546 461 26968.1 1663359F6 1 468 461 26968.1 1663359H1 1 225 462 243103.24 1664686T6 907 1496 462 243103.24 1664686F6 112 673 462 243103.24 1664686H1 112 342 463 469883.1 1670240H1 3910 4101 463 469883.1 1670240F6 3910 4382 463 469883.1 1670240T6 3961 4578 464 903565.14c 1672574T6 43 352 464 903565.14c 1672574F6 2490 3031 464 903565.14c 1672574H1 2830 3031 465 227932.2 1674711H1 4373 4511 466 903691.6 1706278H1 2489 2717 466 903691.6 1706278F6 2489 2870 467 012995.19c 1706278T6 57 561 468 407938.5 1727746T6 7648 8083 468 407938.5 1727746H1 5870 6083 468 407938.5 1727746F6 5870 6174 469 233778.1 1748894F6 271 723 469 233778.1 1748894H1 271 527 469 233778.1 1748894T6 544 792 470 474630.19 1807178F6 2975 3418 470 474630.19 1807178H1 2975 3240 471 217319.7 1808080H1 1303 1574 471 217319.7 1808080F6 1303 1603 471 217319.7 1808080T6 1719 2059 472 1092445.1 1808250F6 2139 2619 472 1092445.1 1808250H1 2363 2628 473 199433.3 1852003F6 51 377 473 199433.3 1852003H1 51 320 473 199433.3 1852003T6 625 761 474 228860.6 1857460F6 92 510 474 228860.6 1857460H1 92 342 475 228860.4c 1857460T6 456 1028 476 410688.3 1891428F6 1 219 476 410688.3 1891428H1 1 279 477 347005.5 1929865F6 6818 7115 477 347005.5 1929865H1 6819 7081 477 347005.5 1929865T6 6818 7074 478 334621.13 1965915R6 329 735 478 334621.13 1965915H1 329 611 478 334621.13 1965915T6 413 975 479 28180.1 1970545F6 411 817 479 28180.1 1970545H1 411 653 479 28180.1 1970545T6 425 814 480 252570.6c 1972687T6 501 1044 480 252570.6c 1972687F6 3 437 480 252570.6c 1972687H1 3 264 481 466402.116 2025345R6 1118 1458 481 466402.116 2025345T6 1118 1419 481 466402.116 2025345H1 1118 1378 482 1096160.26c 2152021F6 961 1348 482 1096160.26c 2152021H1 961 1209 482 1096160.26c 2152021T6 3034 3327 482 1096160.26c 2152021CB1 1 3006 483 899612.2 2188034F6 404 865 483 899612.2 2188034H1 404 715 484 254107.1 2198878F6 1811 2268 484 254107.1 2198878H1 1811 2055 484 254107.1 2198878T6 2597 3123 485 208328.1 2200534F6 2103 2527 485 208328.1 2200534H1 2103 2317 486 130157.2 2295946T6 2207 2554 486 130157.2 2295946R6 1310 1772 486 130157.2 2295946H1 1310 1459 487 985824.2c 2330285T6 1252 1591 487 985824.2c 2330285H1 1252 1507 487 985824.2c 2330285R6 1252 1634 488 233089.1 2350723T6 1921 2022 488 233089.1 2350723F6 1928 2064 489 336256.1 2443920H1 1681 1863 489 336256.1 2443920F6 1681 2077 489 336256.1 2443920T6 2982 3515 490 979616.2 2454756F6 458 830 490 979616.2 2454756H1 458 692 490 979616.2 2454756T6 881 1322 491 351432.3 2457793H1 1332 1537 491 351432.3 2457793F6 1332 1814 491 351432.3 2457793T6 4870 5364 492 83495.1 2499861F6 1 378 492 83495.1 2499861H1 1 255 493 332595.1 2500879F7 1 368 493 332595.1 2500879H1 1 247 493 332595.1 2500879F6 1 543 494 481251.3 2506154T6 967 1069 494 481251.3 2506154F6 1 313 494 481251.3 2506154H1 47 249 495 25612.1 2582525F6 221 371 495 25612.1 2582525H1 221 516 496 96700.1 2788603F6 1 519 496 96700.1 2788603H1 1 231 497 230048.1 2788603T6 142 663 498 073621.3c 2878629F6 1690 1992 499 344582.19 2878629H1 1 216 500 997526.4 2891590F6 1 416 500 997526.4 2891590H1 1 262 501 997526.1 2891590T6 2443 2854
[0181]
Claims
1. A combination comprising a plurality of cDNAs, wherein the cDNAs are selected from SEQ ID NOs:1-501 and the complements of SEQ ID NOs:1-501.
2. The combination of claim 1, wherein each of the cDNAs is downregulated at least two-fold and is selected from SEQ ID NOs:1-56, 87-153, 164-349, 370-414, and 437-501 and the complements of SEQ ID NOs:1-56, 87-153, 164-349, 370-414, and 437-501.
3. The combination of claim 1, wherein each of the cDNAs is upregulated at least two-fold and is selected from SEQ ID NOs:57-86, 154-163, 350-369, and 415-436 and the complements of 57-86, 154-163, 350-369, and 415-436.
4. The combination of claim 1, wherein the cDNAs are immobilized on a substrate.
5. An isolated cDNA selected from SEQ ID NOs: 14, 26, 40, 52, 55, 60, 65, 68, 73, 79, 82, 85, 92, 110, 112, 114, 115, 117, 122, 125, 126, 130, 136, 137, 139, 141, 143, 144, 145, 146, 147, 160, 164, 166, 167, 168, 190, 191, 194, 195, 199, 201, 204, 211, 212, 222, 224, 226, 229, 233, 234, 240, 243, 245, 248, 250, 253, 254, 259, 264, 268, 269, 270, 272, 276, 278, 279, 281, 282, 284, 285, 286, 293, 296, 297, 299, 300, 301, 362, 306, 308, 312, 313, 314, 317, 319, 321, 321, 322, 322, 323, 324, 325, 326, 330, 331, 332, 334, 336, 337, 338, 339, 340, 342, 346, 353, 355, 357, 361, 365, 366, 371, 372, 376, 380, 383, 385, 386, 387, 390, 399, 400, 402, 405, 406, 408, 409, 410, 412, 413, 417, 418, 419, 420, 422, 422, 424, 426, 438, 444, 445, 453, 456, 460, 461, 471, 479, 480, 487, 490, 492, 495, 496, and 497 and the complements thereof.
6. A method for detecting differential expression of one or more cDNAs in a sample containing nucleic acids, the method comprising:
- a) hybridizing the substrate of claim 4 with the nucleic acids, thereby forming one or more hybridization complexes;
- b) detecting hybridization complex formation; and
- c) comparing the complexes so formed with those of a standard, wherein differences between the standard and sample complex formation indicate differential expression of cDNAs in the sample.
7. The method of claim 6, wherein the sample is from prostate.
8. The method of claim 6, wherein differential expression is diagnostic of prostate cancer.
9. A method for screening a plurality of molecules or compounds to identify a ligand which specifically binds the cDNA, the method comprising:
- a) combining the combination of claim 1 with the plurality of molecules or compounds under conditions to allow specific binding; and
- b) detecting specific binding between each cDNA and at least one molecule or compound, thereby identifying a ligand that specifically binds each cDNA.
10. The method of claim 9 wherein the plurality of molecules or compounds are selected from DNA molecules, enhancers, mimetics, RNA molecules, peptide nucleic acids, peptides, repressors and transcription factors.
11. A vector containing the cDNA of claim 5.
12. A host cell containing the vector of claim 11.
13. A method for producing a protein, the method comprising:
- a) culturing the host cell of claim 12 under conditions for expression of protein; and
- b) recovering the protein from the host cell culture.
14. A protein produced by the method of claim 13.
15. A method for using a protein to screen a plurality of molecules or compounds to identify at least one ligand which specifically binds the protein, the method comprising:
- a) combining the protein of claim 14 with the plurality of molecules or compounds under conditions to allow specific binding; and
- b) detecting specific binding between the protein and a molecule or compound, thereby identifying a ligand which specifically binds the protein.
16. The method of claim 15 wherein the plurality of molecules or compounds is selected from agonists, antagonists, antibodies, DNA molecules, mimetics, peptide nucleic acids, peptides, proteins, or pharmaceutical agents, RNA molecules, and small drug molecules.
17. A composition comprising the protein of claim 14 and a pharmaceutical carrier.
18. A method of using a protein to produce and purify an antibody, the method comprising:
- a) immunizing an animal with the protein of claim 14 under conditions to elicit an antibody response;
- b) obtaining a sample containing antibodies;
- c) combining the sample with the protein under conditions to allow specific binding;
- d) recovering the bound protein; and
- e) separating the protein from the antibody, thereby obtaining purified antibody that specifically binds the protein.
19. A purified antibody produced by the method of claim 18.
20. A method of using an antibody to detect prostate cancer, the method comprising:
- a) contacting a sample with the antibody of claim 19 under conditions to form an antibody:protein complex;
- b) detecting antibody:protein complex formation; and
- c) comparing complex formation with standards, wherein complex formation indicates the presence of prostate cancer in the sample.
Type: Application
Filed: May 29, 2002
Publication Date: Oct 9, 2003
Inventors: Mary Faris (Los Angeles, CA), Cecelia I. Pearson (Palo Alto, CA)
Application Number: 10252157
International Classification: C12Q001/68; C07H021/04;
